Methods for treating heterotopic ossification

ABSTRACT

In part, the present disclosure relates methods for treating heterotopic ossification or one or more complications of heterotopic ossification. In particular, the disclosure provided methods for treating heterotopic ossification by administering to a patient in need thereof one or more TGFβ antagonists, optionally in combination with one or more additional active agents or supportive therapies for treating heterotopic ossification. For example, in some embodiments, the disclosure provided methods for treating heterotopic ossifications by administering to a patient in need thereof an effective amount of a TβRII polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication No. 62/628,649, filed on Feb. 9, 2018 and from U.S.Provisional Application No. 62/666,235, filed on May 3, 2018. Theforegoing applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Heterotopic ossification (HO) is the pathologic formation ofextra-skeletal bone in soft tissues. Vanden Bossche and Vanderstraeten,J Rehabil Med 27, 129 (2005). In general, this process occurs in patientpopulations with severe trauma including large-surface area burns,musculoskeletal injury, orthopedic operations, and spinal cord injury,and in patient populations with a genetic disease known asfibrodysplasia ossificans progressiva (FOP). FOP is caused by ahyper-activating mutation in the type I bone morphogenetic protein (BMP)receptor ACVR1, and patients with FOP develop ectopic bone lesions inthe absence of any substantial trauma. The clinical sequela of thesepathologic ectopic bone formations, whether in the setting of trauma orgenetic mutations, include non-healing wounds, chronic pain, and jointimmobility. In the case of FOP, progressive ossification may lead todeath due to loss of thoracic cage compliance.

Treatment options for HO are limited as bone often recurs followingsurgical resection, and some patients may have non-resectable HO due toits sensitive location. The risk of an operation may outweigh thebenefits of excision, especially in the face of recurrence. Thus, thereis a high unmet need for effective therapies for treating HO.

SUMMARY OF THE INVENTION

In part, the present disclosure relates to the discovery that a TGFβantagonist (inhibitor) can be used to treat heterotopic ossification(HO). In particular, the data presented herein shows that a solubleTβRII polypeptide, which binds to TGFβ1 and TGFβ3, is effective atattenuating development of HO anlagen with decreased osseous depositionand marrow space formation in a mouse model of HO. Furthermore,treatment with the TβRII polypeptide significantly reduced the volume ofmature HO in this animal model. While not wishing to be bound to anyparticular mechanism, it is expected that the effects of TβRIIpolypeptides are caused primarily by TGFβ signaling antagonist effect,particularly of TGFβ1 and/or TGFβ3. Therefore, it is expected that otherTGFβ antagonists [e.g., antagonists of the TβRII receptor, antagonistsof one or more TβRII-binding ligand (e.g., TGFβ1, TGFβ2, and TGFβ3),antagonists of one or more TβRII-associated type I receptor (e.g.,ALK5), antagonists of one or more TβRII-associated co-receptor(betaglycan), antagonists of one or more TβRII downstream signalingcomponents (e.g., Smads), or combination of such antagonists] willuseful in the treatment of HO. Such agents are collectively referred toherein as “TGFβ antagonists” or “TGFβ inhibitors”.

In certain aspects, the disclosure relates to methods for treating HOcomprising administering to a patient in need thereof an effectiveamount of a Transforming Growth Factor-β (TGFβ) antagonist. In someembodiments, the methods prevent or reduce the severity and/or durationof one or more complications of HO. For example, treatment with a TGFβantagonist may prevent or reduce the severity and/or duration of one ormore complications of HO selected from the group consisting of: jointcontracture, ankylosis, pain, spasticity, swelling fever, neurovascularcompression, and pressure ulcers. In some embodiments, the methodsrelate to treating HO that is associated with one or more disorders orconditions selected from the group consisting of: spinal cord injury,trauma, brain injuries, burns, fractures, muscle contusion, jointarthroplasty/replacement, hip surgery/replacement, acetabularsurgery/replacement, elbow fracture, fracture of the long bones of thelower leg, combat-related trauma, amputation, neuromuscular blockadeused to manage adult respiratory distress syndrome, nontraumaticmyelopathies. In some embodiments, the disclosure relates to methods oftreating fibrodysplasia ossificans progressiva (FOP) comprisingadministering to a patient in need thereof an effective amount of a TGFβantagonist. For example, the methods prevent or reduce the severityand/or duration of one or more complications of FOP (e.g., heterotopicossification). In some embodiments, the disclosure relates to methods oftreating fibrodysplasia ossificans progressiva (FOP) comprisingadministering to a patient in need thereof an effective amount of a TGFβantagonist. For example, the methods prevent or reduce the severityand/or duration of one or more complications of FOP (e.g., heterotopicossification). In some embodiments, the disclosure relates to methods oftreating fibrous dysplasia comprising administering to a patient in needthereof an effective amount of a TGFβ antagonist. For example, themethods prevent or reduce the severity and/or duration of one or morecomplications of fibrous dysplasia (e.g., heterotopic ossification). Insome embodiments, heterotopic ossification occurs in one or more tissuesselected from the group consisting of: bone, skin, subcutaneous tissue,skeletal muscle, fibrosis tissue adjacent to joints, walls of bloodvessels, and ligaments. Optionally, such methods described hereinfurther comprise administering to the patient an effective amount of oneor more additional active agents or supportive therapies for treatingHO. For example, such additional active agents or supportive therapiesinclude isotretinoin, etidronate with oral corticosteroids, perhexilinemaleate, ALK2 small-molecule inhibitors, palovarotene, retinoic acidreceptor gamma agonists, retinoic acid receptor alpha agonists, activinantibodies (e.g., activin A antibodies such as REGN 2477),allele-specific RNA interference of ALK2, bisphosphonates, radiationtherapy anti-inflammatory agents (e.g., indomethacin, ibuprofen andaspirin), and conservative treatments such as passive range of motionexercises or other mobilization techniques. In some embodiments, theadditional active agent or supportive therapy is treatment with anactivin antagonist. In some embodiments, the activin antagonist is anactivin antibody. In some embodiments, the activin antibody is anactivin A antibody. In some embodiments, TGFβ antagonist is amulti-specific antibody that binds to and inhibits signaling mediated byone or more of TGFβ1, TGFβ2, and TGFβ3 and further binds to and inhibitsactivin (e.g., activin A).

In certain aspects, a TGFβ antagonist, or combination of antagonists, tobe used in accordance with methods and uses described herein is an agentthat inhibits at least TGFβ1 (e.g., a TGFβ1 antagonist). Effects onTGFβ1 inhibition may be determined, for example, using a cell-basedassay including those described herein (e.g., Smad signaling assay).Therefore, in some embodiments, a TGFβ antagonist, or combination ofantagonists, of the disclosure may bind to at least TGFβ1. Ligandbinding activity may be determined, for example, using a bindingaffinity assay including those described herein. In some embodiments, aTGFβ antagonist, or combination of antagonists, of the disclosure bindsto at least TGFβ1 with a K_(D) of at least 1×10⁻⁷ M (e.g., at least1×10⁻⁸ M, at least 1×10⁻⁹ M, at least 1×10⁻¹⁰ M, at least 1×10⁻¹¹ M, orat least 1×10⁻¹² M). As described herein, various TGFβ antagonists thatinhibit TGFβ1 can be used in accordance with the methods and usesdescribed herein including, for example, ligand traps (e.g., TβRIIpolypeptides and variants thereof), antibodies, small molecules,nucleotide sequences, and combinations thereof. In certain embodiments,a TGFβ antagonist, or combination of antagonists, that inhibits TGFβ1may further inhibit one or more of: TGFβ2, TGFβ3, TβRII, ALK5, andbetaglycan. In some embodiments, a TGFβ antagonist, or combination ofantagonists, that inhibits TGFβ1 further inhibits TGFβ3. In someembodiments, a TGFβ antagonist, or combination of antagonists, thatinhibits TGFβ1 does not inhibit or does not substantially inhibit TGFβ2.In some embodiments, a TGFβ antagonist, or combination of antagonists,that inhibits TGFβ1 further inhibits TGFβ3 but does not inhibit or doesnot substantially inhibit TGFβ2.

In certain aspects, a TGFβ antagonist, or combination of antagonists, tobe used in accordance with methods and uses described herein is an agentthat inhibits at least TGFβ2 (e.g., a TGFβ2 antagonist). Effects onTGFβ2 inhibition may be determined, for example, using a cell-basedassay including those described herein (e.g., Smad signaling assay).Therefore, in some embodiments, a TGFβ antagonist, or combination ofantagonists, of the disclosure may bind to at least TGFβ2. Ligandbinding activity may be determined, for example, using a bindingaffinity assay including those described herein. In some embodiments, aTGFβ antagonist, or combination of antagonists, of the disclosure bindsto at least TGFβ2 with a K_(D) of at least 1×10⁻⁷ M (e.g., at least1×10⁻⁸ M, at least 1×10⁻⁹ M, at least 1×10⁻¹⁰ M, at least 1×10⁻¹¹ M, orat least 1×10⁻¹² M). As described herein, various TGFβ antagonists thatinhibit TGFβ2 can be used in accordance with the methods and usesdescribed herein including, for example, ligand traps (e.g., TβRIIpolypeptides and variants thereof), antibodies, small molecules,nucleotide sequences, and combinations thereof. In certain embodiments,a TGFβ antagonist, or combination of antagonists, that inhibits TGFβ2may further inhibit one or more of: TGFβ1, TGFβ3, TβRII, ALK5, andbetaglycan.

In certain aspects, a TGFβ antagonist, or combination of antagonists, tobe used in accordance with methods and uses described herein is an agentthat inhibits at least TGFβ3 (e.g., a TGFβ3 antagonist). Effects onTGFβ3 inhibition may be determined, for example, using a cell-basedassay including those described herein (e.g., Smad signaling assay).Therefore, in some embodiments, a TGFβ antagonist, or combination ofantagonists, of the disclosure may bind to at least TGFβ3. Ligandbinding activity may be determined, for example, using a bindingaffinity assay including those described herein. In some embodiments, aTGFβ antagonist, or combination of antagonists, of the disclosure bindsto at least TGFβ3 with a K_(D) of at least 1×10⁻⁷ M (e.g., at least1×10⁻⁸ M, at least 1×10⁻⁹ M, at least 1×10⁻¹⁰ M, at least 1×10⁻¹¹ M, orat least 1×10⁻¹² M). As described herein, various TGFβ antagonists thatinhibit TGFβ3 can be used in accordance with the methods and usesdescribed herein including, for example, ligand traps (e.g., TβRIIpolypeptides and variants thereof), antibodies, small molecules,nucleotide sequences, and combinations thereof. In certain embodiments,a TGFβ antagonist, or combination of antagonists, that inhibits TGFβ3may further inhibit one or more of: TGFβ1, TGFβ2, TβRII, ALK5, andbetaglycan. In some embodiments, a TGFβ antagonist, or combination ofantagonists, that inhibits TGFβ3 further inhibits TGFβ1. In someembodiments, a TGFβ antagonist, or combination of antagonists, thatinhibits TGFβ3 does not inhibit or does not substantially inhibit TGFβ2.In some embodiments, a TGFβ antagonist, or combination of antagonists,that inhibits TGFβ3 further inhibits TGFβ1 but does not inhibit or doesnot substantially inhibit TGFβ2.

In certain aspects, a TGFβ antagonist, or combination of antagonists, tobe used in accordance with methods and uses described herein is an agentthat inhibits at least TβRII (e.g., a TβRII receptor antagonist).Effects on TβRII inhibition may be determined, for example, using acell-based assay including those described herein (e.g., Smad signalingassay). Therefore, in some embodiments, a TGFβ antagonist, orcombination of antagonists, of the disclosure may bind to at leastTβRII. Ligand binding activity may be determined, for example, using abinding affinity assay including those described herein. In someembodiments, a TGFβ antagonist, or combination of antagonists, of thedisclosure binds to at least TβRII with a K_(D) of at least 1×10⁻⁷ M(e.g., at least 1×10⁻⁸ M, at least 1×10⁻⁹ M, at least 1×10⁻¹⁰ M, atleast 1×10⁻¹¹ M, or at least 1×10⁻¹ M). As described herein, variousTGFβ antagonists that inhibit TβRII can be used in accordance with themethods and uses described herein including, for example, ligand traps(e.g., TβRII polypeptides and variants thereof), antibodies, smallmolecules, nucleotide sequences, and combinations thereof. In certainembodiments, a TGFβ antagonist, or combination of antagonists, thatinhibits TβRII may further inhibit one or more of: TGFβ1, TGFβ2, TGFβ3,ALK5, and betaglycan. In some embodiments, a TGFβ antagonist, orcombination of antagonists, that inhibits TβRII does not inhibit or doesnot substantially inhibit TGFβ2.

In certain aspects, a TGFβ antagonist, or combination of antagonists, tobe used in accordance with methods and uses described herein is an agentthat inhibits at least ALK5 (e.g., na ALK5 antagonist). Effects on ALK5inhibition may be determined, for example, using a cell-based assayincluding those described herein (e.g., Smad signaling assay).Therefore, in some embodiments, a TGFβ antagonist, or combination ofantagonists, of the disclosure may bind to at least ALK5. Ligand bindingactivity may be determined, for example, using a binding affinity assayincluding those described herein. In some embodiments, an ALK5antagonist, or combination of antagonists, of the disclosure binds to atleast ALK5 with a K_(D) of at least 1×10⁻⁷ M (e.g., at least 1×10⁻⁸ M,at least 1×10⁻⁹ M, at least 1×10⁻¹⁰ M, at least 1×10⁻¹¹ M, or at least1×10⁻¹ M). As described herein, various TGFβ antagonists that inhibitALK5 can be used in accordance with the methods and uses describedherein including, for example, ligand traps (e.g., TβRII polypeptidesand variants thereof), antibodies, small molecules, nucleotidesequences, and combinations thereof. In certain embodiments, a TGFβantagonist, or combination of antagonists, that inhibits ALK5 mayfurther inhibit one or more of: TGFβ1, TGFβ2, TGFβ3, TβRII, andbetaglycan. In some embodiments, a TGFβ antagonist, or combination ofantagonists, that inhibits ALK5 does not inhibit or does notsubstantially inhibit TGFβ2.

In certain aspects, a TGFβ antagonist, or combination of antagonists, tobe used in accordance with methods and uses described herein is an agentthat inhibits at least betaglycan (e.g., a betaglycan antagonist).Effects on betaglycan inhibition may be determined, for example, using acell-based assay including those described herein (e.g., Smad signalingassay). Therefore, in some embodiments, a TGFβ antagonist, orcombination of antagonists, of the disclosure may bind to at leastbetaglycan. Ligand binding activity may be determined, for example,using a binding affinity assay including those described herein. In someembodiments, a betaglycan antagonist, or combination of antagonists, ofthe disclosure binds to at least betaglycan with a K_(D) of at least1×10⁻⁷ M (e.g., at least 1×10⁻¹ M, at least 1×10⁻⁹ M, at least 1×10⁻¹⁰M, at least 1×10⁻¹¹ M, or at least 1×10⁻¹² M). As described herein,various TGFβ antagonists that inhibit betaglycan can be used inaccordance with the methods and uses described herein including, forexample, ligand traps (e.g., TβRII polypeptides and variants thereof),antibodies, small molecules, nucleotide sequences, and combinationsthereof. In certain embodiments, a TGFβ antagonist, or combination ofantagonists, that inhibits betaglycan may further inhibit one or more ofTGFβ1, TGFβ2, TGFβ3, TβRII, and ALK5. In some embodiments, a TGFβantagonist, or combination of antagonists, that inhibits betaglycan doesnot inhibit or does not substantially inhibit TGFβ2.

In certain aspects, the disclosure provides TβRII polypeptides and theuse of such TβRII polypeptides as selective antagonists for TGFβ1 and/orTGFβ3. As described herein, polypeptides comprising part or all of theTβRII extracellular domain (ECD), with or without additional mutations,bind to and/or inhibit TGFβ1 and/or TGFβ3 with varying affinities. Thus,in certain aspects, the disclosure provides TβRII polypeptides for usein selectively inhibiting TGFβ superfamily associated disorders.

In part, the disclosure provides TβRII polypeptide fusion proteins andthe use of such fusion proteins as selective antagonists for TGFβ1 orTGFβ3. As described herein, polypeptides comprising part or all of theTβRII extracellular domain (ECD), with or without additional mutations,bind to and/or inhibit TGFβ1 or TGFβ3 with varying affinities. Inparticular, TβRII polypeptides comprising a heterologous portion (e.g.,an Fc immunoglobulin domain) and a linker of at least 10 amino acids inlength (e.g., a linker having the amino acid sequence of SEQ ID NO: 6)are associated with surprisingly superior TGFβ1 and TGFβ3 bindingproperties as compared to TβRII polypeptides having a shorter linker.Thus, in certain aspects, the disclosure provides TβRII polypeptides foruse in selectively inhibiting TGFβ superfamily associated disorders.

In some embodiments, the disclosure provides for a Transforming GrowthFactor-β Receptor II (TβRII) fusion polypeptide comprising: a) anextracellular domain of a TβRII portion; b) a heterologous portion, andc) a linker portion; wherein the linker is at least 10 amino acids inlength; and wherein the TβRII extracellular domain portion comprises anamino acid sequence at least 80% identical to: i) a sequence beginningat any of positions 23 to 35 of SEQ ID NO: 1 and ending at any ofpositions 153 to 159 of SEQ ID NO: 1 or ii) a sequence beginning at anyof positions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178to 184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellulardomain portion comprises an amino acid sequence at least 80% identicalto a sequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion comprises an aminoacid sequence at least 90% identical to a sequence beginning at any ofpositions 23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to159 of SEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence at least 95% identical to asequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion comprises an aminoacid sequence at least 97% identical to a sequence beginning at any ofpositions 23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to159 of SEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence beginning at any of positions23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to 159 ofSEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence at least 80% identical to asequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion comprises an aminoacid sequence at least 90% identical to a sequence beginning at any ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence at least 95% identical to asequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion comprises an aminoacid sequence at least 97% identical to a sequence beginning at any ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence beginning at any of positions23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to 184 ofSEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence at least 80% identical to SEQID NO: 18. In some embodiments, the TβRII extracellular domain portioncomprises an amino acid sequence at least 90% identical to SEQ ID NO:18. In some embodiments, the TβRII extracellular domain portioncomprises an amino acid sequence at least 95% identical to SEQ ID NO:18. In some embodiments, the TβRII extracellular domain portioncomprises an amino acid sequence at least 97% identical to SEQ ID NO:18. In some embodiments, the TβRII extracellular domain portioncomprises the amino acid sequence of SEQ ID NO: 18. In some embodiments,the TβRII extracellular domain portion consists of an amino acidsequence at least 80% identical to a sequence beginning at any ofpositions 23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to159 of SEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion consists of an amino acid sequence at least 90% identical to asequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 95% identical to a sequence beginning at any ofpositions 23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to159 of SEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion consists of an amino acid sequence at least 97% identical to asequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 80% identical to a sequence beginning at any ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion consists of an amino acid sequence at least 90% identical to asequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 95% identical to a sequence beginning at any ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion consists of an amino acid sequence at least 97% identical to asequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 80% identical to SEQ ID NO: 18. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 90% identical to SEQ ID NO: 18. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 95% identical to SEQ ID NO: 18. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 97% identical to SEQ ID NO: 18. In someembodiments, the TβRII extracellular domain portion consists of theamino acid sequence of SEQ ID NO: 18. In some embodiments, thepolypeptide comprises an N-terminal leader sequence. In someembodiments, the N-terminal leader sequence comprises the amino acidsequence of any one of SEQ ID NOs: 22-24. In some embodiments, theN-terminal leader sequence comprises the amino acid sequence of SEQ IDNO: 23. In some embodiments, the heterologous portion is animmunoglobulin Fc domain. In some embodiments, the immunoglobulin Fcdomain is a human immunoglobulin Fc domain. In some embodiments, theheterologous portion comprises an amino acid sequence that is at least80% identical to SEQ ID NO: 49. In some embodiments, the heterologousportion comprises an amino acid sequence that is at least 90% identicalto SEQ ID NO: 49. In some embodiments, the heterologous portioncomprises an amino acid sequence that is at least 95% identical to SEQID NO: 49. In some embodiments, the heterologous portion comprises anamino acid sequence that is at least 97% identical to SEQ ID NO: 49. Insome embodiments, the heterologous portion comprises the amino acidsequence of SEQ ID NO: 49. In some embodiments, the linker is less than25 amino acids in length. In some embodiments, the linker is between 10and 25 amino acids in length. In some embodiments, the linker is between15 and 25 amino acids in length. In some embodiments, the linker isbetween 17 and 22 amino acids in length. In some embodiments, the linkeris 21 amino acids in length. In some embodiments, the linker comprises(GGGGS)n, wherein n=≥2. In some embodiments, the linker comprises(GGGGS)n, wherein n=≥3. In some embodiments, the linker comprises(GGGGS)n, wherein n=≥4. In some embodiments, the linker comprises(GGGGS)_(n), wherein n≠≥5. In some embodiments, the linker comprises theamino acid sequence of SEQ ID NO: 21. In some embodiments, the linkercomprises the amino acid sequence of any one of SEQ ID NOs: 4-7. In someembodiments, the linker comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the polypeptide comprises an amino acid sequencethat is at least 80% identical to the amino acid sequence of SEQ ID NO:11. In some embodiments, the polypeptide comprises an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 11. In some embodiments, the polypeptide comprises an aminoacid sequence that is at least 95% identical to the amino acid sequenceof SEQ ID NO: 11. In some embodiments, the polypeptide comprises theamino acid sequence of SEQ ID NO: 11. In some embodiments, thepolypeptide comprises an amino acid sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO: 13. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 13. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 13. In someembodiments, the polypeptide comprises the amino acid sequence of SEQ IDNO: 13. In some embodiments, the polypeptide comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO: 63. In some embodiments, the polypeptide comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 63. In some embodiments, the polypeptide comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 63. In some embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 63. In some embodiments,the polypeptide comprises an amino acid sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO: 48. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 48. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 48. In someembodiments, the polypeptide comprises the amino acid sequence of SEQ IDNO: 48. In some embodiments, the polypeptide comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO: 65. In some embodiments, the polypeptide comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 65. In some embodiments, the polypeptide comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 65. In some embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 65. In some embodiments,the polypeptide comprises an amino acid sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the polypeptide comprises the amino acid sequence of SEQ IDNO: 68. In some embodiments, the polypeptide comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO: 15. In some embodiments, the polypeptide comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 15. In some embodiments, the polypeptide comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 15. In some embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 15. In some embodiments,the TβRII polypeptide does not include amino acids 185-592 of SEQ ID NO:2. In some embodiments, the TβRII polypeptide does not include aminoacids 1-22 of SEQ ID NO: 2. In some embodiments, the polypeptideconsists of or consists essentially of: a) a TβRII polypeptide portioncomprising an amino acid sequence that is at least 85%, 90%, 95%, 97%,or 99% identical to the amino acid sequence of SEQ ID NO: 18 and no morethan 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linkerportion comprising an amino acid sequence that is at least 85%, 90%,95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 6and no more than 5, 4, 3, 2 or 1 additional amino acids; c) aheterologous portion comprising an amino acid sequence that is at least85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQID NO: 49 and no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additionalamino acids; and d) optionally a leader sequence (e.g., SEQ ID NO: 23).In some embodiments, the polypeptide consists of or consists essentiallyof: a) a TβRII polypeptide portion comprising the amino acid sequence ofSEQ ID NO: 18 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1additional amino acids; b) a linker portion comprising the amino acidsequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1 additionalamino acids; c) a heterologous portion comprising the amino acidsequence of SEQ ID NO: 49 and no more than 25, 20, 15, 10, 5, 4, 3, 2,or 1 additional amino acids; and d) optionally a leader sequence (e.g.,SEQ ID NO: 23). In some embodiments, the polypeptide comprises: a) anextracellular domain of a TβRII portion; wherein the extracellulardomain comprises an amino acid sequence that is at least 85%, 90%, 95%,97%, or 99% identical to the sequence of SEQ ID NO: 18; b) aheterologous portion, wherein the heterologous portion comprises anamino acid sequence that is at least 85%, 90%, 95%, 97%, or 99%identical to the sequence of SEQ ID NO: 49; and c) a linker portionconnecting the extracellular domain and the heterologous portion;wherein the linker comprises an amino acid sequence that is at least85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQID NO: 6. In some embodiments, the polypeptide comprises: a) anextracellular domain of a TβRII portion; wherein the extracellulardomain comprises the amino acid sequence of SEQ ID NO: 18; b) aheterologous portion, wherein the heterologous portion comprises theamino acid sequence of SEQ ID NO: 49; and c) a linker portion connectingthe extracellular domain and the heterologous portion; wherein thelinker comprises the amino acid sequence of SEQ ID NO: 6. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence ofSEQ ID NO: 48. In some embodiments, the polypeptide comprises the aminoacid sequence of SEQ ID NO: 48. In some embodiments, the polypeptidedoes not include a leader sequence, or wherein the leader sequence hasbeen removed. In some embodiments, the polypeptide includes one or moremodified amino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent.In some embodiments, the polypeptide is glycosylated. In someembodiments, the polypeptide has a glycosylation pattern characteristicof expression of the polypeptide in CHO cells. In some embodiments, thepolypeptide binds human TGFβ1 with an equilibrium dissociation constant(K_(D)) less than 100 pM. In some embodiments, the polypeptide bindshuman TGFβ1 with an equilibrium dissociation constant (K_(D)) less than75 pM. In some embodiments, the polypeptide binds human TGFβ3 with anequilibrium dissociation constant (K_(D)) less than 60 pM. In someembodiments, the polypeptide binds human TGFβ3 with an equilibriumdissociation constant (K_(D)) less than 50 pM. In some embodiments, thepolypeptide inhibits TGFβ1 with an IC50 of less than 1.0 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ1 with an IC50 of less than 0.25 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ1 with an IC50 of less than 0.1 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ1 with an IC50 of less than 0.05 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ3 with an IC50 of less than 0.3 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ3 with an IC50 of less than 0.1 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ3 with an IC50 of less than 0.05 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ3 with an IC50 of less than 0.04 nM, asdetermined using a reporter gene assay. In some embodiments, thereporter gene assay is a CAGA reporter assay.

In some embodiments, the disclosure provides for a homodimer comprisingany two of the polypeptides disclosed herein.

In some embodiments, the disclosure provides for an isolatedpolynucleotide comprising a coding sequence for any of the polypeptidesdisclosed herein. In some embodiments, the disclosure provides for arecombinant polynucleotide comprising a promoter sequence operablylinked to any of the polynucleotides disclosed herein. In someembodiments, the polynucleotide comprises a nucleotide sequence that isat least 80%, 85%, 90%, 95%, 97% or 100% identical to any one of SEQ IDNOs: 10, 12, 14, 46 or 47.

In some embodiments, the disclosure provides for a cell transformed withany of the polynucleotides disclosed herein. In some embodiments, cellis a mammalian cell. In some embodiments, the cell is a CHO cell or ahuman cell.

In some embodiments, the disclosure provides for a pharmaceuticalpreparation comprising pharmaceutically acceptable excipient and any ofthe polypeptides disclosed herein or any of the homodimers disclosedherein.

In certain aspects, a TGFβ antagonist is an antibody, or combination ofantibodies. In certain aspects, the antibody binds to at least TβRII. Insome embodiments a TGFβ antagonist antibody that binds to TβRII inhibitsTβRII signaling, optionally as measured in a cell-based assay such asthose described herein. In some embodiments, a TGFβ antagonist antibodythat binds to TβRII inhibits one or more TGF-beta superfamily ligands,TGFβ superfamily type I receptors, or TGFβ superfamily co-receptors frombinding to TβRII. In some embodiments, a TGFβ antagonist antibody thatbinds to TβRII inhibits one or more TGF-beta superfamily ligands frombinding to TβRII selected from the group consisting of: TGFβ1, TGFβ2,and TGFβ3. In certain aspects, the antibody binds to at least ALK5. Insome embodiments a TGFβ antagonist antibody that binds to ALK5 inhibitsALK5 signaling, optionally as measured in a cell-based assay such asthose described herein. In some embodiments, a TGFβ antagonist antibodythat binds to ALK5 inhibits one or more TGF-beta superfamily ligands,TGFβ superfamily type II receptors, or TGFβ superfamily co-receptorsfrom binding to ALK5. In some embodiments a TGFβ antagonist antibodythat binds to ALK5 inhibits one or more TGF-beta superfamily ligandsfrom binding to ALK5 selected from the group consisting of: TGFβ1,TGFβ2, and TGFβ3. In certain aspects, the antibody binds to at leastbetaglycan. In some embodiments a TGFβ antagonist antibody that binds tobetaglycan inhibits betaglycan signaling, optionally as measured in acell-based assay such as those described herein. In some embodiments, aTGFβ antagonist antibody that binds to betaglycan inhibits one or moreTGF-beta superfamily ligands, TGFβ superfamily type I receptors, or TGFβsuperfamily type II receptors from binding to betaglycan. In someembodiments a TGFβ antagonist antibody that binds to betaglycan inhibitsone or more TGF-beta superfamily ligands from binding to betaglycanselected from the group consisting of: TGFβ1, TGFβ2, and TGFβ3. Incertain aspects, a TGFβ antagonist antibody binds to at least TGFβ1. Insome embodiments, a TGFβ antagonist antibody that binds to TGFβ1inhibits TβRII signaling, optionally as measured in a cell-based assaysuch as those described herein. In some embodiments, a TGFβ antagonistantibody that binds to TGFβ1 inhibits TGFβ1-TβRII, TGFβ1-ALK5, and/orTGFβ1-betaglycan binding. In certain aspects, a TGFβ antagonist antibodybinds to at least TGFβ2. In some embodiments, a TGFβ antagonist antibodythat binds to TGFβ2 inhibits TβRII signaling, optionally as measured ina cell-based assay such as those described herein. In some embodiments,a TGFβ antagonist antibody that binds to TGFβ2 inhibits TGFβ2-TβRII,TGFβ1-ALK5, and/or TGFβ1-betaglycan binding. In certain embodiments, aTGFβ antagonist antibody binds to at least TGFβ3. In some embodiments, aTGFβ antagonist antibody that binds to TGFβ3 inhibits TβRII signaling,optionally as measured in a cell-based assay such as those describedherein. In some embodiments, a TGFβ antagonist antibody that binds toTGFβ3 inhibits TGFβ3-TβRII, TGFβ1-ALK5, and/or TGFβ1-betaglycan binding.In some embodiments, a TGFβ antagonist antibody is a multispecificantibody, or a combination of multispecific antibodies, inhibitssignaling in a cell-based assay of one or more of: TGFβ1, TGFβ2, TGFβ3,TβRII, ALK5, and betaglycan. In some embodiments, antibody is a chimericantibody, a humanized antibody, or a human antibody. In someembodiments, the antibody is a single-chain antibody, an F(ab′)2fragment, a single-chain diabody, a tandem single-chain Fv fragment, atandem single-chain diabody, or a fusion protein comprising asingle-chain diabody and at least a portion of an immunoglobulinheavy-chain constant region.

In certain aspects, a TGFβ antagonist is a small molecule inhibitor orcombination of small molecule inhibitors. In some embodiments, a TGFβantagonist small molecule inhibitor is an inhibitor of at least TβRII.In some embodiments, a TGFβ antagonist small molecule inhibitor is aninhibitor of at least ALK5. In some embodiments, a TGFβ antagonist smallmolecule inhibitor is an inhibitor of at least betaglycan. In someembodiments, a TGFβ antagonist small molecule inhibitor is an inhibitorof at least TGFβ1. In some embodiments, a TGFβ antagonist small moleculeinhibitor is an inhibitor of at least TGFβ2. In some embodiments, a TGFβantagonist small molecule inhibitor is an inhibitor of at least TGFβ3.

In certain aspects, a TGFβ antagonist is a nucleic acid inhibitor orcombination of nucleic acid inhibitors. In some embodiments, a TGFβantagonist nucleic acid inhibitor is an inhibitor of at least TβRII. Insome embodiments, a TGFβ antagonist nucleic acid inhibitor is aninhibitor of at least ALK5. In some embodiments, a TGFβ antagonistnucleic acid inhibitor is an inhibitor of at least betaglycan. In someembodiments, a TGFβ antagonist nucleic acid inhibitor is an inhibitor ofat least TGFβ1. In some embodiments, a TGFβ antagonist nucleic acidinhibitor is an inhibitor of at least TGFβ2. In some embodiments, a TGFβantagonist nucleic acid inhibitor is an inhibitor of at least TGFβ3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of native precursor for the B(short) isoform of human TGFβ receptor type II (hTβRII) (NP_003233.4).Solid underline indicates the mature extracellular domain (ECD)(residues 23-159), and double underline indicates valine that isreplaced in the A (long) isoform. Dotted underline denotes leader(residues 1-22).

FIG. 2 shows the amino acid sequence of native precursor for the A(long) isoform of human TβRII (NP_001020018.1). Solid underlineindicates the mature ECD (residues 23-184), and double underlineindicates the splice-generated isoleucine substitution. Dotted underlinedenotes leader (residues 1-22).

FIG. 3 shows a comparison of the linker sequences of five differentTβRII constructs.

FIGS. 4A and 4B show in tabular form the binding affinity between TGFβ1and TGFβ3 and one of several different TβRII-Fc fusion proteinconstructs.

FIGS. 5A and 5C graph the results from reporter gene assays testing theaffinity of TGFβ1 for one of several different TβRII-Fc fusion proteinconstructs. FIGS. 5B and 5D graph the results from reporter gene assaystesting the affinity of the TGFβ3 for one of several different TβRII-Fcfusion protein constructs. FIGS. 5E and 5F provide IC₅₀ data from thesesame experiments in tabular form.

FIGS. 6A, 6B, and 6C show the effects of mTβRII-mFc in an in vitroinflammation model as well as an animal model of heterotopicossification. FIG. 6A shows that mTβRII-mFc can abrogate TGFβ1 secretionby regenerative M2 macrophages in an inflammatory environment in vitro.FIG. 6B shows that treatment with mTβRII-mFc decreases osseousdeposition and marrow space formation in an animal model of traumaticheterotopic ossification. FIG. 6C shows that treatment with mTβRII-mFcsignificantly decreases the volume of mature HO in an animal model oftraumatic heterotopic ossification.

FIG. 7 shows multiple sequence alignment of Fc domains from human IgGisotypes using Clustal 2.1. Hinge regions are indicated by dottedunderline. Double underline indicates examples of positions engineeredin IgG1 Fc to promote asymmetric chain pairing and the correspondingpositions with respect to other isotypes IgG2, IgG3 and IgG4.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

Proteins described herein are the human forms, unless otherwisespecified. NCBI references for the proteins are as follows: human TβRIIisoform A (hTβRI_(long)), NP_001020018.1 and human TβRII isoform B(hTβRII_(short)), (NP_003233.4). Sequences of native TβRII proteins fromhuman are set forth in FIGS. 1 and 2. In some embodiments, the TβRIIproteins are from non-human animals, such as a mouse, rat, cow ormonkey.

The TGFβ superfamily contains a variety of growth factors that sharecommon sequence elements and structural motifs. These proteins are knownto exert biological effects on a large variety of cell types in bothvertebrates and invertebrates. Members of the superfamily performimportant functions during embryonic development in pattern formationand tissue specification and can influence a variety of differentiationprocesses, including adipogenesis, myogenesis, chondrogenesis,cardiogenesis, hematopoiesis, neurogenesis, and epithelial celldifferentiation. By manipulating the activity of a member of the TGFβfamily, it is often possible to cause significant physiological changesin an organism. For example, the Piedmontese and Belgian Blue cattlebreeds carry a loss-of-function mutation in the GDF8 (also calledmyostatin) gene that causes a marked increase in muscle mass. Grobet etal., Nat Genet. 1997, 17(1):71-4. Similarly, in humans, inactive allelesof GDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

TGFβ signals are mediated by heteromeric complexes of type I (e.g. TβRI)and type II (e.g. TβRII) serine/threonine kinase receptors, whichphosphorylate and activate downstream SMAD proteins upon ligandstimulation (Massague, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178). Thesetype I and type II receptors are transmembrane proteins, composed of aligand-binding extracellular domain with cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine specificity. Type I receptors are essential forsignaling; and type II receptors are required for binding ligands andfor expression of type I receptors. Type I and II receptors form astable complex after ligand binding, resulting in phosphorylation oftype I receptors by type II receptors. TGFβ has three mammalianisoforms, TGFβ1, TGFβ2 and TGFβ3, each with distinct functions in vivo.The binding of TGFβs to TβRII is a crucial step in initiating activationof the TGFβ signaling pathway, leading to phosphorylation of SMAD2, andtranslocation of the activated SMAD2/SMAD4 complex to the nucleus tomodulate gene expression.

Thus, in certain aspects, the disclosure provides TβRII polypeptides asantagonists of TGFβ1 or TGFβ3 for use in treating various TGFβ1- orTGFβ3-associated disorders. While not wishing to be bound to anyparticular mechanism of action, it is expected that such polypeptidesact by binding to TGFβ1 or TGFβ3 and inhibiting the ability of theseligands to form ternary signaling complexes.

The disclosure provides for fusion proteins comprising TβRIIpolypeptides and a heterologous portion (e.g., an Fc portion). Inparticular embodiments, the TβRII portion and the heterologous portionare fused by means of a linker. As described in greater detail below,the disclosure demonstrates that TβRII-Fc fusion proteins comprisinglinkers of certain lengths (e.g., a linker having 21 amino acids) weresurprisingly able to bind TGFβ-1 and TGFβ-3 with stronger affinity thanTβRII-Fc fusion proteins having a linker of only four amino acids.

As demonstrated herein, a soluble TβRII polypeptide, which binds toTGFβ1 and TGFβ3, is effective at substantially attenuating developmentof heterotopic ossification (HO) anlagen with decreased osseousdeposition and marrow space formation in a mouse model of HO.Furthermore, treatment with the TβRII polypeptide significantly reducedthe volume of mature HO in this animal model. While not wishing to bebound to any particular mechanism, it is expected that the effects ofTβRII polypeptides are caused primarily by TGFβ signaling antagonisteffect, particularly of TGFβ1 and/or TGFβ3. Regardless of the mechanism,it is apparent from the data presented herein the TGFβ signalingantagonists do reduce the severity of osseous deposition in HO. Itshould be noted that heterotopic ossification is dynamic, which changesdepending on a balance of factors that increase HO and factors thatdecrease HO. HO can be decreased by increasing factors that reduce HO;decreasing factors that increase HO; or both. Therefore, the termdecreasing (reducing) HO refers to the observable physical changes inosseous deposition and are intended to be neutral as to the mechanismsby which the changes occur.

The animal model for HO that was used in the studies descried herein areconsidered to be predicative of efficacy in humans, and therefore, thisdisclosure provides methods for using TGFβ signaling antagonist to treatHO particularly preventing or reducing the severity or duration of oneor more complications of HO, in humans. As disclosed herein, the termTGFβ antagonist refers a variety of agents that may be used toantagonize TGFβ signaling including, for example, antagonists thatinhibit one or more TGFβ ligands [e.g., TGFβ1, TGFβ2, and/or TGFβ3];antagonists that inhibit one or more TGFβ-interacting type I-, type II-,or co-receptor (e.g., TβRII, ALK5 and betaglycan); and antagonists thatinhibit one or more downstream signaling components (e.g., Smad proteinssuch as Smads 2 and 3). TGFβ antagonists to be used in accordance withthe methods and uses of the disclosure include a variety of forms, forexample, ligand traps (e.g., soluble TβRII polypeptides and betaglycanpolypeptides), antibody antagonists (e.g., antibodies that inhibit oneor more of TGFβ1, TGFβ2, TGFβ3, TβRII, and betaglycan), small moleculeantagonists [e.g., small molecules that inhibit one or more of TGFβ1,TGFβ2, TGFβ3, TβRII, betaglycan, and one or more Smad proteins (e.g.,Smads 2 and 3)], and nucleotide antagonists [e.g., nucleotide sequencesthat inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII, betaglycan, andone or more Smad proteins (e.g., Smads 2 and 3)].

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions. The term “sequencesimilarity,” in all its grammatical forms, refers to the degree ofidentity or correspondence between nucleic acid or amino acid sequencesthat may or may not share a common evolutionary origin. However, incommon usage and in the instant application, the term “homologous,” whenmodified with an adverb such as “highly,” may refer to sequencesimilarity and may or may not relate to a common evolutionary origin.

“Percent (%) sequence identity” or “percent (%) identical” with respectto a reference polypeptide (or nucleotide) sequence is defined as thepercentage of amino acid residues (or nucleic acids) in a candidatesequence that are identical to the amino acid residues (or nucleicacids) in the reference polypeptide (nucleotide) sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for aligning sequences, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid (nucleic acid) sequence identity values are generated using thesequence comparison computer program ALIGN-2. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc., and thesource code has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087. The ALIGN-2 program is publiclyavailable from Genentech, Inc., South San Francisco, Calif., or may becompiled from the source code. The ALIGN-2 program should be compiledfor use on a UNIX operating system, including digital UNIX V4.0D. Allsequence comparison parameters are set by the ALIGN-2 program and do notvary.

“Agonize”, in all its grammatical forms, refers to the process ofactivating a protein and/or gene (e.g., by activating or amplifying thatprotein's gene expression or by inducing an inactive protein to enter anactive state) or increasing a protein's and/or gene's activity.

“Antagonize”, in all its grammatical forms, refers to the process ofinhibiting a protein and/or gene (e.g., by inhibiting or decreasing thatprotein's gene expression or by inducing an active protein to enter aninactive state) or decreasing a protein's and/or gene's activity.

The terms “about” and “approximately” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges.

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers. As used herein, the term “comprises”also encompasses the use of the narrower terms “consisting” and“consisting essentially of.”

The term “consisting essentially of” is limited to the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristics of the invention(s) disclosed herein.

The term “appreciable affinity” as used herein means binding with adissociation constant (K_(D)) of less than 50 nM.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength. The chain may be linear or branched, it may comprise modifiedamino acids, and/or may be interrupted by non-amino acids. The termsalso encompass an amino acid chain that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that the polypeptides can occur as single chains orassociated chains.

2. TGFβ Antagonists

As disclosed herein, the term TGFβ antagonist refers a variety of agentsthat may be used to antagonize TGFβ-mediated signaling including, forexample, antagonists that inhibit one or more TGFβ ligands [e.g., TGFβ1,TGFβ2, and/or TGFβ3]; antagonists that inhibit one or moreTGFβ-interacting type I-, type II-, or co-receptor (e.g., TβRII, ALK5and betaglycan); and antagonists that inhibit one or more downstreamsignaling components (e.g., Smad proteins such as Smads 2 and 3). TGFβantagonists to be used in accordance with the methods and uses of thedisclosure include a variety of forms, for example, ligand traps (e.g.,soluble TβRII polypeptides and betaglycan polypeptides), antibodyantagonists (e.g., antibodies that inhibit one or more of TGFβ1, TGFβ2,TGFβ3, TβRII, and betaglycan), small molecule antagonists [e.g., smallmolecules that inhibit one or more of TGFβ1, TGFβ2, TGFβ3, TβRII,betaglycan, and one or more Smad proteins (e.g., Smads 2 and 3)], andnucleotide antagonists [e.g., nucleotide sequences that inhibit one ormore of TGFβ1, TGFβ2, TGFβ3, TβRII, betaglycan, and one or more Smadproteins (e.g., Smads 2 and 3)].

A. TβRII and Betaglycan Polypeptides

Naturally occurring TβRII proteins are transmembrane proteins, with aportion of the protein positioned outside the cell (the extracellularportion) and a portion of the protein positioned inside the cell (theintracellular portion). Aspects of the present disclosure encompassvariant TβRII polypeptides comprising mutations within the extracellulardomain and/or truncated portions of the extracellular domain of TβRII.As described above, human TβRII occurs naturally in at least twoisoforms—A (long) and B (short)—generated by alternative splicing in theextracellular domain (ECD) (FIGS. 1 and 2 and SEQ ID NOS: 1 and 2). SEQID NO: 27, which corresponds to residues 23-159 of SEQ ID NO: 1, depictsthe native full-length extracellular domain of the short isoform ofTβRII. SEQ ID NO: 18, which corresponds to residues 23-184 of SEQ ID NO:2, depicts the native full-length extracellular domain of the longisoform of TβRII. Unless noted otherwise, amino acid position numberingwith regard to variants based on the TβRII short and long isoformsrefers to the corresponding position in the native precursors, SEQ IDNO: 1 and SEQ ID NO: 2, respectively.

In certain embodiments, the disclosure provides variant TβRIIpolypeptides. A TβRII polypeptide of the disclosure may bind to andinhibit the function of a TGFβ superfamily member, such as but notlimited to, TGFβ1 or TGFβ3. TβRII polypeptides may include a polypeptideconsisting of, or comprising, an amino acid sequence at least 70%identical, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a truncated ECDdomain of a naturally occurring TβRII polypeptide, whose C-terminusoccurs at any of amino acids 153-159 of SEQ ID NO: 1. TβRII polypeptidesmay include a polypeptide consisting of, or comprising, an amino acidsequence at least 70% identical, and optionally at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto a truncated ECD domain of a naturally occurring TβRII polypeptide,whose C-terminus occurs at any of amino acids 178-184 of SEQ ID NO: 2.In particular embodiments, the TβRII polypeptides comprise an amino acidsequence at least 70% identical, and optionally at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the amino acid sequence of SEQ ID NO: 18. Optionally, a TβRIIpolypeptide does not include more than 5 consecutive amino acids, ormore than 10, 20, 30, 40, 50, 52, 60, 70, 80, 90, 100, 150 or 200 ormore consecutive amino acids from a sequence consisting of amino acids160-567 of SEQ ID NO: 1 or from a sequence consisting of amino acids185-592 of SEQ ID NO: 2. In some embodiments, the TβRII polypeptide doesnot include amino acids 160-567 of SEQ ID NO: 1. In some embodiments,the TβRII polypeptide does not include amino acids 1-22 of SEQ ID NO: 1.In some embodiments, the TβRII polypeptide does not include amino acids1-22 and 160-567 of SEQ ID NO: 1. In some embodiments, the TβRIIpolypeptide does not include amino acids 185-592 of SEQ ID NO: 2. Insome embodiments, the TβRII polypeptide does not include amino acids1-22 of SEQ ID NO: 2. In some embodiments, the TβRII polypeptide doesnot include amino acids 1-22 and 185-592 of SEQ ID NO: 2. Theunprocessed TβRII polypeptide may either include or exclude any signalsequence, as well as any sequence N-terminal to the signal sequence. Aselaborated herein, the N-terminus of the mature (processed) TβRIIpolypeptide may occur at any of amino acids 23-35 of SEQ ID NO: 1 or23-60 of SEQ ID NO: 2. Examples of mature TβRII polypeptides include,but are not limited to, amino acids 23-159 of SEQ ID NO: 1 (set forth inSEQ ID NO: 27), amino acids 29-159 of SEQ ID NO: 1 (set forth in SEQ IDNO: 28), amino acids 35-159 of SEQ ID NO: 1 (set forth in SEQ ID NO:29), amino acids 23-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 30),amino acids 29-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 31), aminoacids 35-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 32), amino acids23-184 of SEQ ID NO: 2 (set forth in SEQ ID NO: 18), amino acids 29-184of SEQ ID NO: 2 (set forth in SEQ ID NO: 33), amino acids 60-184 of SEQID NO: 2 (set forth in SEQ ID NO: 29), amino acids 23-178 of SEQ ID NO:2 (set forth in SEQ ID NO: 34), amino acids 29-178 of SEQ ID NO: 2 (setforth in SEQ ID NO: 35), and amino acids 60-178 of SEQ ID NO: 2 (setforth in SEQ ID NO: 32). It will be understood by one of skill in theart that corresponding variants based on the long isoform of TβRII willinclude nucleotide sequences encoding the 25-amino acid insertion alongwith a conservative Val-Ile substitution at the flanking positionC-terminal to the insertion. The TβRII polypeptides accordingly mayinclude isolated extracellular portions of TβRII polypeptides, includingboth the short and the long isoforms, variants thereof (includingvariants that comprise, for example, no more than 2, 3, 4, 5, 10, 15,20, 25, 30, or 35 amino acid substitutions in the sequence correspondingto amino acids 23-159 of SEQ ID NO: 1 or amino acids 23-184 of SEQ IDNO: 2), fragments thereof, and fusion proteins comprising any of theforegoing, but in each case preferably any of the foregoing TβRIIpolypeptides will retain substantial affinity for at least one of, orboth of, TGFβ1 or TGFβ3. Generally, a TβRII polypeptide will be designedto be soluble in aqueous solutions at biologically relevanttemperatures, pH levels, and osmolarity.

In some embodiments, the variant TβRII polypeptides of the disclosurecomprise one or more mutations in the extracellular domain that conferan altered ligand binding profile. A TβRII polypeptide may include one,two, five or more alterations in the amino acid sequence relative to thecorresponding portion of a naturally occurring TβRII polypeptide. Insome embodiments, the mutation results in a substitution, insertion, ordeletion at the position corresponding to position 70 of SEQ ID NO: 1.In some embodiments, the mutation results in a substitution, insertion,or deletion at the position corresponding to position 110 of SEQ IDNO: 1. Examples include, but are not limited to, an N to D substitutionor a D to K substitution in the positions corresponding to positions 70and 110, respectively, of SEQ ID NO: 1. Examples of such variant TβRIIpolypeptides include, but are not limited to, the sequences set forth inSEQ ID NOs: 36-39. A TβRII polypeptide may comprise a polypeptide orportion thereof that is encoded by any one of SEQ ID NOs: 8, 10, 12, 14,16, 46 or 47, or silent variants thereof or nucleic acids that hybridizeto the complement thereof under stringent hybridization conditions. Inparticular embodiments, a TβRII polypeptide may comprise a polypeptideor portion thereof that is encoded by any one of SEQ ID NO: 12, orsilent variants thereof or nucleic acids that hybridize to thecomplement thereof under stringent hybridization conditions.

In some embodiments, the variant TβRII polypeptides of the disclosurefurther comprise an insertion of 36 amino acids (SEQ ID NO: 41) betweenthe pair of glutamate residues (positions 151 and 152 of SEQ ID NO: 1,or positions 176 and 177 of SEQ ID NO: 2) located near the C-terminus ofthe human TβRII ECD, as occurs naturally in the human TβRII isoform C(Konrad et al., BMC Genomics 8:318, 2007).

It has been demonstrated that TβRII polypeptides can be modified toselectively antagonize TβRII ligands. The N70 residue represents apotential glycosylation site. In some embodiments, the TβRIIpolypeptides are aglycosylated. In some embodiments, the TβRIIpolypeptides are aglycosylated or have reduced glycosylation at positionAsn157. In some embodiments, the TβRII polypeptides are aglycosylated orhave reduced glycosylation at position Asn73.

In certain embodiments, a TβRII polypeptide binds to TGFβ1, and theTβRII polypeptide does not show substantial binding to TGFβ3. In certainembodiments, a TβRII polypeptide binds to TGFβ3, and the TβRIIpolypeptide does not show substantial binding to TGFβ1. Binding may beassessed using purified proteins in solution or in a surface plasmonresonance system, such as a Biacore™ system.

In certain embodiments, a TβRII polypeptide inhibits TGFβ1 cellularsignaling, and the TβRII polypeptide has an intermediate or limitedinhibitory effect on TGFβ3 signaling. In certain embodiments, a TβRIIpolypeptide inhibits TGFβ3 cellular signaling, and the TβRII polypeptidehas an intermediate or limited inhibitory effect on TGFβ1 signaling.Inhibitory effect on cell signaling can be assayed by methods known inthe art.

Taken together, an active portion of a TβRII polypeptide may compriseamino acid sequences 23-153, 23-154, 23-155, 23-156, 23-157, or 23-158of SEQ ID NO: 1, as well as variants of these sequences starting at anyof amino acids 24-35 of SEQ ID NO: 1. Similarly, an active portion of aTβRII polypeptide may comprise amino acid sequences 23-178, 23-179,23-180, 23-181, 23-182, or 23-183 of SEQ ID NO: 2, as well as variantsof these sequences starting at any of amino acids 24-60 of SEQ ID NO: 2.Exemplary TβRII polypeptides comprise amino acid sequences 29-159,35-159, 23-153, 29-153 and 35-153 of SEQ ID NO: 1 or amino acidsequences 29-184, 60-184, 23-178, 29-178 and 60-178 of SEQ ID NO: 2.Variants within these ranges are also contemplated, particularly thosehaving at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity to the corresponding portion of SEQ ID NO: 1or SEQ ID NO: 2. A TβRII polypeptide may be selected that does notinclude the sequence consisting of amino acids 160-567 of SEQ ID NO: 1or amino acids 185-592 of SEQ ID NO: 2. In particular embodiments, theTβRII polypeptides comprise an amino acid sequence at least 70%identical, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acidsequence of SEQ ID NO: 18.

The term “betaglycan polypeptide” includes polypeptides comprising anynaturally occurring betaglycan protein (encoded by TGFBR3 or one of itsnonhuman orthologs) as well as any variants thereof (including mutants,fragments, fusions, and peptidomimetic forms) that retain a usefulactivity.

The human betaglycan isoform A precursor protein sequence (NCBI Ref SeqNP_003234.2) is as follows:

(SEQ ID NO: 50)  1 MTSHYVIAIF ALMSSCLATA GPEPGALCEL SPVSASHPVQ ALMESFTVLS GCASRGTTGL 61 PQEVHVLNLR TAGQGPGQLQ REVTLHLNPI SSVHIHHKSV VFLLNSPHPL VWHLKTERLA121 TGVSRLFLVS EGSVVQFSSA NFSLTAETEE RNFPHGNEHL LNWARKEYGA VTSFTELKIA181 RNIYIKVGED QVFPPKCNIG KNFLSLNYLA EYLQPKAAEG CVMSSQPQNE EVHIIELITP241 NSNPYSAFQV DITIDIRPSQ EDLEVVKNLI LILKCKKSVN WVIKSFDVKG SLKIIAPNSI301 GFGKESERSM TMTKSIRDDI PSTQGNLVKW ALDNGYSPIT SYTMAPVANR FHLRLENN

E 361 EMGDEEVHTI PPELRILLDP GALPALQNPP IRGGEGQNGG LPFPFPDISR RVWNEEGEDG421 LPRPKDPVIP SIQLFPGLRE PEEVQGSVDI ALSVKCDNEK MIVAVEKDSF QASGYSGMDV481 TLLDPTCKAK MNGTHFVLES PLNGCGTRPR WSALDGVVYY NSIVIQVPAL GDSSGWPDGY541 EDLESGDNGF PGDMDEGDAS LFTRPEIVVF NCSLQQVRNP SSFQEQPHGN ITFNMELYNT601 DLFLVPSQGV FSVPENGHVY VEVSVTKAEQ ELGFAIQTCF ISPYSNPDRM SHYTIIENIC661 PKDESVEFYS PKRVHFPIPQ ADMDKERFSF VFKPVFNTSL LFLQCELTLC TKMEKHPQKL721 PKCVPPDEAC TSLDASIIWA MMQNKKTFTK PLAVIHHEAE SKEKGPSMEE PNPISPPIFH

841 QSTPCSSSST A

The signal peptide is indicated by single underline, the extracellulardomain is indicated in bold font, and the transmembrane domain isindicated by dotted underline. This isoform differs from betaglycanisoform B by insertion of a single alanine indicated above by doubleunderline.

A processed betaglycan isoform A polypeptide sequence is as follows:

(SEQ ID NO: 51) GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFPHGNEHLLNWARKEYGAVISFTELKIARNIYIKVGEDQVFPPKCNIGKNELSLNYLAEYLQPKAAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKCKKSVNWVIKSFDVKGSLKIIAPNSIGEGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNAEEMGDEEVHTIPPELRILLDPGALPALQNPPIRGGEGQNGGLPFPFPDISRRVWNEEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSVDIALSVKCDNEKMIVAVEKDSFQASGYSGMDVILLDPICKAKMNGTHFVLESPLNGCGTRPRWSALDGVVYYNSIVIQVPALGDSSGWPDGYEDLESGDNGFPGDMDEGDASLFTRPEIVVENCSLQQVRNPSSFQEQPHGNITFNMELYNTDLFLVPSQGVESVPENGHVYVEVSVIKAEQELGFAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHFPIPQADMDKKRFSFVFKPVFNISLLFLQCELTLCIKMEKHPQKLPKCVPPDEACTSLDASIIWAMMQNKKTFTKPLAVIHHEAESKEKGPSMKE PNPISPPIFHGLDILTV

A nucleic acid sequence encoding the unprocessed precursor protein ofhuman betaglycan isoform A is shown below (SEQ ID NO: 52), correspondingto nucleotides 516-3068 of NCBI Reference Sequence NM_003243.4. Thesignal sequence is indicated by solid underline and the transmembraneregion by dotted underline.

(SEQ ID NO: 52)ATGACTTCCCATTATGTGATTGCCATCTTTGCCCTGATGAGCTCCTGTTTAGCCACTGCAGGTCCAGAGCCTGGTGCACTGTGTGAACTGTCACCTGTCAGTGCCTCCCATCCTGTCCAGGCCTTGATGGAGAGCTTCACTGTTTTGTCAGGCTGTGCCAGCAGAGGCACAACTGGGCTGCCACAGGAGGTGCATGTCCTGAATCTCCGCACTGCAGGCCAGGGGCCTGGCCAGCTACAGAGAGAGGTCACACTTCACCTGAATCCCATCTCCTCAGTCCACATCCACCACAAGTCTGTTGTGTTCCTGCTCAACTCCCCACACCCCCTGGTGTGGCATCTGAAGACAGAGAGACTTGCCACTGGGGTCTCCAGACTGTTTTTGGTGTCTGAGGGTTCTGTGGTCCAGTTTTCATCAGCAAACTTCTCCTTGACAGCAGAAACAGAAGAAAGGAACTTCCCCCATGGAAATGAACATCTGTTAAATTGGGCCCGAAAAGAGTATGGAGCAGTTACTTCATTCACCGAACTCAAGATAGCAAGAAACATTTATATTAAAGTGGGGGAAGATCAAGTGTTCCCTCCAAAGTGCAACATAGGGAAGAATTTTCTCTCACTCAATTACCTTGCTGAGTACCTTCAACCCAAAGCAGCAGAAGGGTGTGTGATGTCCAGCCAGCCCCAGAATGAGGAAGTACACATCATCGAGCTAATCACCCCCAACTCTAACCCCTACAGTGCTTTCCAGGTGGATATAACAATTGATATAAGACCTTCTCAAGAGGATCTTGAAGTGGTCAAAAATCTCATCCTGATCTTGAAGTGCAAAAAGTCTGTCAACTGGGTGATCAAATCTTTTGATGTTAAGGGAAGCCTGAAAATTATTGCTCCTAACAGTATTGGCTTTGGAAAAGAGAGTGAAAGATCTATGACAATGACCAAATCAATAAGAGATGACATTCCTTCAACCCAAGGGAATCTGGTGAAGTGGGCTTTGGACAATGGCTATAGTCCAATAACTTCATACACAATGGCTCCTGTGGCTAATAGATTTCATCTTCGGCTTGAAAATAATGCAGAGGAGATGGGAGATGAGGAAGTCCACACTATTCCTCCTGAGCTACGGATCCTGCTGGACCCTGGTGCCCTGCCTGCCCTGCAGAACCCGCCCATCCGGGGAGGGGAAGGCCAAAATGGAGGCCTTCCGTTTCCTTTCCCAGATATTTCCAGGAGAGTCTGGAATGAAGAGGGAGAAGATGGGCTCCCTCGGCCAAAGGACCCTGTCATTCCCAGCATACAACTGTTTCCTGGTCTCAGAGAGCCAGAAGAGGTGCAAGGGAGCGTGGATATTGCCCTGTCTGTCAAATGTGACAATGAGAAGATGATCGTGGCTGTAGAAAAAGATTCTTTTCAGGCCAGTGGCTACTCGGGGATGGACGTCACCCTGTTGGATCCTACCTGCAAGGCCAAGATGAATGGCACACACTTTGTTTTGGAGTCTCCTCTGAATGGCTGCGGTACTCGGCCCCGGTGGTCAGCCCTTGATGGTGTGGTCTACTATAACTCCATTGTGATACAGGTTCCAGCCCTTGGGGACAGTAGTGGTTGGCCAGATGGTTATGAAGATCTGGAGTCAGGTGATAATGGATTTCCGGGAGATATGGATGAAGGAGATGCTTCCCTGTTCACCCGACCTGAAATCGTGGTGTTTAATTGCAGCCTTCAGCAGGTGAGGAACCCCAGCAGCTTCCAGGAACAGCCCCACGGAAACATCACCTTCAACATGGAGCTATACAACACTGACCTCTTTTTGGTGCCCTCCCAGGGCGTCTTCTCTGTGCCAGAGAATGGACACGTTTATGTTGAGGTATCTGTTACTAAGGCTGAACAAGAACTGGGATTTGCCATCCAAACGTGCTTTATCTCTCCATATTCGAACCCTGATAGGATGTCTCATTACACCATTATTGAGAATATTTGTCCTAAAGATGAATCTGTGAAATTCTACAGTCCCAAGAGAGTGCACTTTCCTATCCCGCAAGCTGACATGGATAAGAAGCGATTCAGCTTTGTCTTCAAGCCTGTCTTCAACACCTCACTGCTCTTTCTACAGTGTGAGCTGACGCTGTGTACGAAGATGGAGAAGCACCCCCAGAAGTTGCCTAAGTGTGTGCCTCCTGACGAAGCCTGCACCTCGCTGGACGCCTCGATAATCTGGGCCATGATGCAGAATAAGAAGACGTTCACTAAGCCCCTTGCTGTGATCCACCATGAAGCAGAATCTAAAGAAAAAGGTCCAAGCATGAAGGAACCAAATCCAATTTCT

CCAGCCTCGGAAAACAGCAGTGCTGCCCACAGCATCGGCAGCACGCAGAGCACGCCTTGCTCCAGCAGCAGCACGGCC

A nucleic acid sequence encoding a processed extracellular domain ofbetaglycan isoform A is shown below (SEQ ID NO: 53):

(SEQ ID NO: 53) GGTCCAGAGCCTGGTGCACTGTGTGAACTGTCACCTGTCAGTGCCTCCCATCCTGTCCAGGCCTTGATGGAGAGCTTCACTGTTTTGTCAGGCTGTGCCAGCAGAGGCACAACTGGGCTGCCACAGGAGGTGCATGTCCTGAATCTCCGCACTGCAGGCCAGGGGCCTGGCCAGCTACAGAGAGAGGTCACACTTCACCTGAATCCCATCTCCTCAGTCCACATCCACCACAAGTCTGTTGTGTTCCTGCTCAACTCCCCACACCCCCTGGTGTGGCATCTGAAGACAGAGAGACTTGCCACTGGGGTCTCCAGACTGTTTTTGGTGTCTGAGGGTTCTGTGGTCCAGTTTTCATCAGCAAACTTCTCCTTGACAGCAGAAACAGAAGAAAGGAACTTCCCCCATGGAAATGAACATCTGTTAAATTGGGCCCGAAAAGAGTATGGAGCAGTTACTTCATTCACCGAACTCAAGATAGCAAGAAACATTTATATTAAAGTGGGGGAAGATCAAGTGTTCCCTCCAAAGTGCAACATAGGGAAGAATTTTCTCTCACTCAATTACCTTGCTGAGTACCTTCAACCCAAAGCAGCAGAAGGGTGTGTGATGTCCAGCCAGCCCCAGAATGAGGAAGTACACATCATCGAGCTAATCACCCCCAACTCTAACCCCTACAGTGCTTTCCAGGTGGATATAACAATTGATATAAGACCTTCTCAAGAGGATCTTGAAGTGGTCAAAAATCTCATCCTGATCTTGAAGTGCAAAAAGTCTGTCAACTGGGTGATCAAATCTTTTGATGTTAAGGGAAGCCTGAAAATTATTGCTCCTAACAGTATTGGCTTTGGAAAAGAGAGTGAAAGATCTATGACAATGACCAAATCAATAAGAGATGACATTCCTTCAACCCAAGGGAATCTGGTGAAGTGGGCTTTGGACAATGGCTATAGTCCAATAACTTCATACACAATGGCTCCTGTGGCTAATAGATTTCATCTTCGGCTTGAAAATAATGCAGAGGAGATGGGAGATGAGGAAGTCCACACTATTCCTCCTGAGCTACGGATCCTGCTGGACCCTGGTGCCCTGCCTGCCCTGCAGAACCCGCCCATCCGGGGAGGGGAAGGCCAAAATGGAGGCCTTCCGTTTCCTTTCCCAGATATTTCCAGGAGAGTCTGGAATGAAGAGGGAGAAGATGGGCTCCCTCGGCCAAAGGACCCTGTCATTCCCAGCATACAACTGTTTCCTGGTCTCAGAGAGCCAGAAGAGGTGCAAGGGAGCGTGGATATTGCCCTGTCTGTCAAATGTGACAATGAGAAGATGATCGTGGCTGTAGAAAAAGATTCTTTTCAGGCCAGTGGCTACTCGGGGATGGACGTCACCCTGTTGGATCCTACCTGCAAGGCCAAGATGAATGGCACACACTTTGTTTTGGAGTCTCCTCTGAATGGCTGCGGTACTCGGCCCCGGTGGTCAGCCCTTGATGGTGTGGTCTACTATAACTCCATTGTGATACAGGTTCCAGCCCTTGGGGACAGTAGTGGTTGGCCAGATGGTTATGAAGATCTGGAGTCAGGTGATAATGGATTTCCGGGAGATATGGATGAAGGAGATGCTTCCCTGTTCACCCGACCTGAAATCGTGGTGTTTAATTGCAGCCTTCAGCAGGTGAGGAACCCCAGCAGCTTCCAGGAACAGCCCCACGGAAACATCACCTTCAACATGGAGCTATACAACACTGACCTCTTTTTGGTGCCCTCCCAGGGCGTCTTCTCTGTGCCAGAGAATGGACACGTTTATGTTGAGGTATCTGTTACTAAGGCTGAACAAGAACTGGGATTTGCCATCCAAACGTGCTTTATCTCTCCATATTCGAACCCTGATAGGATGTCTCATTACACCATTATTGAGAATATTTGTCCTAAAGATGAATCTGTGAAATTCTACAGTCCCAAGAGAGTGCACTTTCCTATCCCGCAAGCTGACATGGATAAGAAGCGATTCAGCTTTGTCTTCAAGCCTGTCTTCAACACCTCACTGCTCTTTCTACAGTGTGAGCTGACGCTGTGTACGAAGATGGAGAAGCACCCCCAGAAGTTGCCTAAGTGTGTGCCTCCTGACGAAGCCTGCACCTCGCTGGACGCCTCGATAATCTGGGCCATGATGCAGAATAAGAAGACGTTCACTAAGCCCCTTGCTGTGATCCACCATGAAGCAGAATCTAAAGAAAAAGGTCCAAGCATGAAGGAACCAAATCCAATTTCTCCACCAATTTTCCATGGTCTGGACACCCTAACCGT G

A human betaglycan isoform B precursor protein sequence (NCBI Ref SeqNP_001182612.1) is as follows:

(SEQ ID NO: 54)  1 MTSHYVIAIF ALMSSCLATA GPEPGALCEL SPVSASHPVQ ALMESFTVLS GCASRGTTGL 61 PQEVHVLNLR TAGQGPGQLQ REVTLHLNPI SSVHIHHKSV VFLLNSPHPL VWHLKTERLA121 TGVSRLFLVS EGSVVQFSSA NFSLTAETEE RNFPHGNEHL LNWARKEYGA VTSFTELKIA181 RNIYIKVGED QVFPPKCNIG KNFLSLNYLA EYLQPKAAEG CVMSSQPQNE EVHIIELITP241 NSNPYSAFQV DITIDIRPSQ EDLEVVKNLI LILKCKKSVN WVIKSFDVKG SLKIIAPNSI301 GFGKESERSM TMTKSIRDDI PSTQGNLVKW ALDNGYSPIT SYTMAPVANR FHLRLENNEE361 MGDEEVHTIP PELRILLDPG ALPALQNPPI RGGEGQNGGL PFPFPDISRR VWNEEGEDGL421 PRPKDPVIPS IQLFPGLREP EEVQGSVDIA LSVKCDNEKM IVAVEKDSFQ ASGYSGMDVT481 LLDPTCKAKM NGTHFVLESP LNGCGTRPRW SALDGVVYYN SIVIQVPALG DSSGWPDGYE541 DLESGDNGFP GDMDEGDASL FTRPEIVVFN CSLQQVRNPS SFQEQPHGNI TFNMELYNTD601 LFLVPSQGVF SVPENGHVYV EVSVTKAEQE LGFAIQTCFI SPYSNPDRMS HYTIIENICP661 KDESVKFYSP KRVHFPIPQA DMDKERFSFV FKPVFNTSLL FLQCELTLCT KMEKHPQKLP721 KCVPPDEACT SLDASIIWAM MQNKKTFTKP LAVIHHEAES KEKGPSMKEP NPISPPIFHG

841 STPCSSSSTA

The signal peptide is indicated by single underline, the extracellulardomain is indicated in bold font, and the transmembrane domain isindicated by

A processed betaglycan isoform B polypeptide sequence is as follows:

(SEQ ID NO: 55) GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKCKKSVNWVIKSFDVKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEMGDEEVHTIPPELRILLDPGALPALQNPPIRGGEGQNGGLPFPFPDISRRVWNEEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSVDIALSVKCDNEKMIVAVEKDSFQASGYSGMDVTLLDPTCKAKMNGTHFVLESPLNGCGTRPRWSALDGVVYYNSIVIQVPALGDSSGWPDGYEDLESGDNGFPGDMDEGDASLFTRPEIVVFNCSLQQVRNPSSFQEQPHGNITFNMELYNTDLFLVPSQGVFSVPENGHVYVEVSVTKAEQELGFAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHFPIPQADMDKKRFSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKLPKCVPPDEACTSLDASIIWAMMQNKKTFTKPLAVIHHEAESKEKGPSMKEP NPISPPIFHGLDTLTV

A nucleic acid sequence encoding the unprocessed precursor protein ofhuman betaglycan isoform B is shown below (SEQ ID NO: 56), correspondingto nucleotides 516-3065 of NCBI Reference Sequence NM_001195683.1. Thesignal sequence is indicated by solid underline and the transmembraneregion by

(SEQ ID NO: 56)ATGACTTCCCATTATGTGATTGCCATCTTTGCCCTGATGAGCTCCTGTTTAGCCACTGCAGGTCCAGAGCCTGGTGCACTGTGTGAACTGTCACCTGTCAGTGCCTCCCATCCTGTCCAGGCCTTGATGGAGAGCTTCACTGTTTTGTCAGGCTGTGCCAGCAGAGGCACAACTGGGCTGCCACAGGAGGTGCATGTCCTGAATCTCCGCACTGCAGGCCAGGGGCCTGGCCAGCTACAGAGAGAGGTCACACTTCACCTGAATCCCATCTCCTCAGTCCACATCCACCACAAGTCTGTTGTGTTCCTGCTCAACTCCCCACACCCCCTGGTGTGGCATCTGAAGACAGAGAGACTTGCCACTGGGGTCTCCAGACTGTTTTTGGTGTCTGAGGGTTCTGTGGTCCAGTTTTCATCAGCAAACTTCTCCTTGACAGCAGAAACAGAAGAAAGGAACTTCCCCCATGGAAATGAACATCTGTTAAATTGGGCCCGAAAAGAGTATGGAGCAGTTACTTCATTCACCGAACTCAAGATAGCAAGAAACATTTATATTAAAGTGGGGGAAGATCAAGTGTTCCCTCCAAAGTGCAACATAGGGAAGAATTTTCTCTCACTCAATTACCTTGCTGAGTACCTTCAACCCAAAGCAGCAGAAGGGTGTGTGATGTCCAGCCAGCCCCAGAATGAGGAAGTACACATCATCGAGCTAATCACCCCCAACTCTAACCCCTACAGTGCTTTCCAGGTGGATATAACAATTGATATAAGACCTTCTCAAGAGGATCTTGAAGTGGTCAAAAATCTCATCCTGATCTTGAAGTGCAAAAAGTCTGTCAACTGGGTGATCAAATCTTTTGATGTTAAGGGAAGCCTGAAAATTATTGCTCCTAACAGTATTGGCTTTGGAAAAGAGAGTGAAAGATCTATGACAATGACCAAATCAATAAGAGATGACATTCCTTCAACCCAAGGGAATCTGGTGAAGTGGGCTTTGGACAATGGCTATAGTCCAATAACTTCATACACAATGGCTCCTGTGGCTAATAGATTTCATCTTCGGCTTGAAAATAATGAGGAGATGGGAGATGAGGAAGTCCACACTATTCCTCCTGAGCTACGGATCCTGCTGGACCCTGGTGCCCTGCCTGCCCTGCAGAACCCGCCCATCCGGGGAGGGGAAGGCCAAAATGGAGGCCTTCCGTTTCCTTTCCCAGATATTTCCAGGAGAGTCTGGAATGAAGAGGGAGAAGATGGGCTCCCTCGGCCAAAGGACCCTGTCATTCCCAGCATACAACTGTTTCCTGGTCTCAGAGAGCCAGAAGAGGTGCAAGGGAGCGTGGATATTGCCCTGTCTGTCAAATGTGACAATGAGAAGATGATCGTGGCTGTAGAAAAAGATTCTTTTCAGGCCAGTGGCTACTCGGGGATGGACGTCACCCTGTTGGATCCTACCTGCAAGGCCAAGATGAATGGCACACACTTTGTTTTGGAGTCTCCTCTGAATGGCTGCGGTACTCGGCCCCGGTGGTCAGCCCTTGATGGTGTGGTCTACTATAACTCCATTGTGATACAGGTTCCAGCCCTTGGGGACAGTAGTGGTTGGCCAGATGGTTATGAAGATCTGGAGTCAGGTGATAATGGATTTCCGGGAGATATGGATGAAGGAGATGCTTCCCTGTTCACCCGACCTGAAATCGTGGTGTTTAATTGCAGCCTTCAGCAGGTGAGGAACCCCAGCAGCTTCCAGGAACAGCCCCACGGAAACATCACCTTCAACATGGAGCTATACAACACTGACCTCTTTTTGGTGCCCTCCCAGGGCGTCTTCTCTGTGCCAGAGAATGGACACGTTTATGTTGAGGTATCTGTTACTAAGGCTGAACAAGAACTGGGATTTGCCATCCAAACGTGCTTTATCTCTCCATATTCGAACCCTGATAGGATGTCTCATTACACCATTATTGAGAATATTTGTCCTAAAGATGAATCTGTGAAATTCTACAGTCCCAAGAGAGTGCACTTTCCTATCCCGCAAGCTGACATGGATAAGAAGCGATTCAGCTTTGTCTTCAAGCCTGTCTTCAACACCTCACTGCTCTTTCTACAGTGTGAGCTGACGCTGTGTACGAAGATGGAGAAGCACCCCCAGAAGTTGCCTAAGTGTGTGCCTCCTGACGAAGCCTGCACCTCGCTGGACGCCTCGATAATCTGGGCCATGATGCAGAATAAGAAGACGTTCACTAAGCCCCTTGCTGTGATCCACCATGAAGCAGAATCTAAAGAAAAAGGTCCAAGCATGAAGGAACCAAATCCAATTTCTCCA

GCCTCGGAAAACAGCAGTGCTGCCCACAGCATCGGCAGCACGCAGAGCACGCCTTGCTCCAGCAGCAGCACGGCC

A nucleic acid sequence encoding a processed extracellular domain ofbetaglycan isoform B is shown below (SEQ ID NO: 57):

(SEQ ID NO: 57) GGTCCAGAGCCTGGTGCACTGTGTGAACTGTCACCTGTCAGTGCCTCCCATCCTGTCCAGGCCTTGATGGAGAGCTTCACTGTTTTGTCAGGCTGTGCCAGCAGAGGCACAACTGGGCTGCCACAGGAGGTGCATGTCCTGAATCTCCGCACTGCAGGCCAGGGGCCTGGCCAGCTACAGAGAGAGGTCACACTTCACCTGAATCCCATCTCCTCAGTCCACATCCACCACAAGTCTGTTGTGTTCCTGCTCAACTCCCCACACCCCCTGGTGTGGCATCTGAAGACAGAGAGACTTGCCACTGGGGTCTCCAGACTGTTTTTGGTGTCTGAGGGTTCTGTGGTCCAGTTTTCATCAGCAAACTTCTCCTTGACAGCAGAAACAGAAGAAAGGAACTTCCCCCATGGAAATGAACATCTGTTAAATTGGGCCCGAAAAGAGTATGGAGCAGTTACTTCATTCACCGAACTCAAGATAGCAAGAAACATTTATATTAAAGTGGGGGAAGATCAAGTGTTCCCTCCAAAGTGCAACATAGGGAAGAATTTTCTCTCACTCAATTACCTTGCTGAGTACCTTCAACCCAAAGCAGCAGAAGGGTGTGTGATGTCCAGCCAGCCCCAGAATGAGGAAGTACACATCATCGAGCTAATCACCCCCAACTCTAACCCCTACAGTGCTTTCCAGGTGGATATAACAATTGATATAAGACCTTCTCAAGAGGATCTTGAAGTGGTCAAAAATCTCATCCTGATCTTGAAGTGCAAAAAGTCTGTCAACTGGGTGATCAAATCTTTTGATGTTAAGGGAAGCCTGAAAATTATTGCTCCTAACAGTATTGGCTTTGGAAAAGAGAGTGAAAGATCTATGACAATGACCAAATCAATAAGAGATGACATTCCTTCAACCCAAGGGAATCTGGTGAAGTGGGCTTTGGACAATGGCTATAGTCCAATAACTTCATACACAATGGCTCCTGTGGCTAATAGATTTCATCTTCGGCTTGAAAATAATGAGGAGATGGGAGATGAGGAAGTCCACACTATTCCTCCTGAGCTACGGATCCTGCTGGACCCTGGTGCCCTGCCTGCCCTGCAGAACCCGCCCATCCGGGGAGGGGAAGGCCAAAATGGAGGCCTTCCGTTTCCTTTCCCAGATATTTCCAGGAGAGTCTGGAATGAAGAGGGAGAAGATGGGCTCCCTCGGCCAAAGGACCCTGTCATTCCCAGCATACAACTGTTTCCTGGTCTCAGAGAGCCAGAAGAGGTGCAAGGGAGCGTGGATATTGCCCTGTCTGTCAAATGTGACAATGAGAAGATGATCGTGGCTGTAGAAAAAGATTCTTTTCAGGCCAGTGGCTACTCGGGGATGGACGTCACCCTGTTGGATCCTACCTGCAAGGCCAAGATGAATGGCACACACTTTGTTTTGGAGTCTCCTCTGAATGGCTGCGGTACTCGGCCCCGGTGGTCAGCCCTTGATGGTGTGGTCTACTATAACTCCATTGTGATACAGGTTCCAGCCCTTGGGGACAGTAGTGGTTGGCCAGATGGTTATGAAGATCTGGAGTCAGGTGATAATGGATTTCCGGGAGATATGGATGAAGGAGATGCTTCCCTGTTCACCCGACCTGAAATCGTGGTGTTTAATTGCAGCCTTCAGCAGGTGAGGAACCCCAGCAGCTTCCAGGAACAGCCCCACGGAAACATCACCTTCAACATGGAGCTATACAACACTGACCTCTTTTTGGTGCCCTCCCAGGGCGTCTTCTCTGTGCCAGAGAATGGACACGTTTATGTTGAGGTATCTGTTACTAAGGCTGAACAAGAACTGGGATTTGCCATCCAAACGTGCTTTATCTCTCCATATTCGAACCCTGATAGGATGTCTCATTACACCATTATTGAGAATATTTGTCCTAAAGATGAATCTGTGAAATTCTACAGTCCCAAGAGAGTGCACTTTCCTATCCCGCAAGCTGACATGGATAAGAAGCGATTCAGCTTTGTCTTCAAGCCTGTCTTCAACACCTCACTGCTCTTTCTACAGTGTGAGCTGACGCTGTGTACGAAGATGGAGAAGCACCCCCAGAAGTTGCCTAAGTGTGTGCCTCCTGACGAAGCCTGCACCTCGCTGGACGCCTCGATAATCTGGGCCATGATGCAGAATAAGAAGACGTTCACTAAGCCCCTTGCTGTGATCCACCATGAAGCAGAATCTAAAGAAAAAGGTCCAAGCATGAAGGAACCAAATCCAATTTCTCCACCAATTTTCCATGGTCTGGACACCCTAACCGTG

In certain embodiments, the disclosure relates to betaglycanpolypeptide, which includes fragments, functional variants, and modifiedforms thereof. Preferably, betaglycan polypeptides for use in accordancewith inventions of the disclosure are soluble (e.g., comprise anextracellular domain of betaglycan). In other preferred embodiments,betaglycan polypeptides for use in accordance with the inventions of thedisclosure bind to and/or inhibit (antagonize) activity (e.g., Smadsignaling) of one or more TGF-beta superfamily ligands. In someembodiments, betaglycan polypeptide comprise an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs:50, 51, 54, or 55. In some embodiments, betaglycan polypeptides comprisean amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolypeptide that begins at any one of amino acids of 21-28 of SEQ ID NO:50, and ends at any one of amino acids 381-787 of SEQ ID NO: 50. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 21-381 of SEQ ID NO: 50. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 21-787 of SEQ ID NO: 50. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 28-381 of SEQ ID NO: 50. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 28-787 of SEQ ID NO: 50. In someembodiments, polypeptides of the disclosure comprise of at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 21-781 of SEQ ID NO: 50. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 28-781 of SEQ ID NO: 50. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolypeptide that begins at any one of amino acids of 21-28 of SEQ ID NO:54, and ends at any one of amino acids 380-786 of SEQ ID NO: 54. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 21-380 of SEQ ID NO: 54. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 21-786 of SEQ ID NO: 54. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 28-380 of SEQ ID NO: 54. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 28-786 of SEQ ID NO: 54. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 21-780 of SEQ ID NO: 54. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 28-780 of SEQ ID NO: 54. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolypeptide that begins at any one of amino acids of 21-28 of SEQ ID NO:50, and ends at any one of amino acids 730-787 of SEQ ID NO: 50. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 21-787 of SEQ ID NO: 50. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 28-730 of SEQ ID NO: 50. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolypeptide that begins at any one of amino acids of 21-28 of SEQ ID NO:50, and ends at any one of amino acids 730-787 of SEQ ID NO: 50. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 21-787 of SEQ ID NO: 50. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 28-730 of SEQ ID NO: 50. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolypeptide that begins at any one of amino acids of 21-28 (e.g., aminoacid residues of SEQ ID NO: 54, and ends at any one of amino acids730-787 of SEQ ID NO: 54. In some embodiments, polypeptides of thedisclosure comprise at least one betaglycan polypeptide that is at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids of 21-786 of SEQ ID NO: 54. In someembodiments, polypeptides of the disclosure comprise at least onebetaglycan polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsof 28-729 of SEQ ID NO: 54.

In certain aspects, the present disclosure relates to heteromultimersthat comprise an ALK5 polypeptide. As used herein, the term “ALK5”refers to a family of activin receptor-like kinase-5 proteins from anyspecies and variants derived from such ALK4 proteins by mutagenesis orother modification. Reference to ALK5 herein is understood to be areference to any one of the currently identified forms. Members of theALK5 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK5 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK5 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity.

The human ALK5 precursor protein, isoform 1 sequence (NCBI Ref SeqNP_004603.1) is as follows:

(SEQ ID NO: 69) 1 MEAAVAAPRP RLLLLVLAAA AAAA AALLPG ATALQCFCHLCTKDNFTCVT DGLCFVSVTE  61 TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS KTGSVTTTYCCNQDHCNKIE LPTTVKSSPG  121 LGPVELAAVI AGPVCFVCIS LMLMVYICHN RTVIHHRVPNEEDPSLDRPF ISEGTTLKDL  181 IYDMTTSGSG SGLPLLVQRT IARTIVLQES IGKGRFGEVWRGKWRGEEVA VKIFSSREER  241 SWFREAEIYQ TVMLRHENIL GFIAADNKDN GTWTQLWLVSDYHEHGSLFD YLNRYTVTVE  301 GMIKLALSTA SGLAHLHMEI VGTQGKPAIA HRDLKSKNILVKKNGTCCIA DLGLAVRHDS  361 ATDTIDIAPN HRVGTKRYMA PEVLDDSINM KHFESFKRADIYAMGLVFWE IARRCSIGGI  421 HEDYQLPYYD LVPSDPSVEE MRKVVCEQKL RPNIPNRWQSCEALRVMAKI MRECWYANGA  481 ARLTALRIKK TLSQLSQQEG IKM

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

A processed extracellular ALK5 polypeptide sequence is as follows:

(SEQ ID NO: 70) AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPV EL

A nucleic acid sequence encoding the ALK5 precursor protein is shownbelow (SEQ ID NO: 32), corresponding to nucleotides 77-1585 of GenbankReference Sequence NM_004612.2. The signal sequence is underlined andthe extracellular domain is indicated in bold font.

(SEQ ID NO: 71) ATGGAGGCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCCTCGTGCTGGCGGCGGCGGCGGCGGCGGCG GCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGCTGGACCAGTGTGCTTCGTCTGCATCTCACTCATGTTGATGGTCTATATCTGCCACAACCGCACTGTCATTCACCATCGAGTGCCAAATGAAGAGGACCCTTCATTAGATCGCCCTTTTATTTCAGAGGGTACTACGTTGAAAGACTTAATTTATGATATGACAACGTCAGGTTCTGGCTCAGGTTTACCATTGCTTGTTCAGAGAACAATTGCGAGAACTATTGTGTTACAAGAAAGCATTGGCAAAGGTCGATTTGGAGAAGTTTGGAGAGGAAAGTGGCGGGGAGAAGAAGTTGCTGTTAAGATATTCTCCTCTAGAGAAGAACGTTCGTGGTTCCGTGAGGCAGAGATTTATCAAACTGTAATGTTACGTCATGAAAACATCCTGGGATTTATAGCAGCAGACAATAAAGACAATGGTACTTGGACTCAGCTCTGGTTGGTGTCAGATTATCATGAGCATGGATCCCTTTTTGATTACTTAAACAGATACACAGTTACTGTGGAAGGAATGATAAAACTTGCTCTGTCCACGGCGAGCGGTCTTGCCCATCTTCACATGGAGATTGTTGGTACCCAAGGAAAGCCAGCCATTGCTCATAGAGATTTGAAATCAAAGAATATCTTGGTAAAGAAGAATGGAACTTGCTGTATTGCAGACTTAGGACTGGCAGTAAGACATGATTCAGCCACAGATACCATTGATATTGCTCCAAACCACAGAGTGGGAACAAAAAGGTACATGGCCCCTGAAGTTCTCGATGATTCCATAAATATGAAACATTTTGAATCCTTCAAACGTGCTGACATCTATGCAATGGGCTTAGTATTCTGGGAAATTGCTCGACGATGTTCCATTGGTGGAATTCATGAAGATTACCAACTGCCTTATTATGATCTTGTACCTTCTGACCCATCAGTTGAAGAAATGAGAAAAGTTGTTTGTGAACAGAAGTTAAGGCCAAATATCCCAAACAGATGGCAGAGCTGTGAAGCCTTGAGAGTAATGGCTAAAATTATGAGAGAATGTTGGTATGCCAATGGAGCAGCTAGGCTTACAGCATTGCGGATTAAGAAAACATTATCGCAACTCAGTCAACAGGAAGGC ATCAAAATG

A nucleic acid sequence encoding an extracellular human ALK5 polypeptideis as follows:

(SEQ ID NO: 72) GCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTG TGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGT ATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTG TGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGTAAAGTCATC ACCTGGCCTTGGTCCTGTGGAACTG

An alternative isoform of the human ALK5 precursor protein sequence,isoform 2 (NCBI Ref Seq XP_005252207.1), is as follows:

(SEQ ID NO: 73)   1 MEAAVAAPRP RLLLLVLAAA AAAA AALLPG    ATALQCFCHL CTKDNFTCVT DGLCFVSVTE 61 TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS    KTGSVTTTYC CNQDHCNKIE LPTTGPFSVK121 SSPGLGPVEL AAVIAGPVCF VCISLMLMVY    ICHNRTVIHH RVPNEEDPSL DRPFISEGTT181 LKDLIYDMTT SGSGSGLPLL VQRTIARTIV    LQESIGKGRF GEVWRGKWRG EEVAVKIFSS241 REERSWFREA EIYQTVMLRH ENILGFIAAD    NKDNGTWTQL WLVSDYHEHG SLFDYLNRYT301 VTVEGMIKLA LSTASGLAHL HMEIVGTQGK    PAIAHRDLKS KNILVKKNGT CCIADLGLAV361 RHDSATDTID IAPNHRVGTK RYMAPEVLDD    SINMKHFESF KRADIYAMGL VFWEIARRCS 421 IGGIHEDYQL PYYDLVPSDP SVEEMRKWC    EQKLRPNIPN RWQSCEALRV MAKIMRECWY 481 ANGAARLTAL RIKKTLSQLS QQEGIKM

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

A processed extracellular ALK5 polypeptide sequence (isoform 2) is asfollows:

(SEQ ID NO: 74) AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVT TTYCCNQDHCNKIELPTTGPFSVKSSPGLGPVEL

A nucleic acid sequence encoding human ALK5 precursor protein (isoform2) is shown below (SEQ ID NO: 75), corresponding to nucleotides 77-1597of Genbank Reference Sequence XM_005252150.1. The signal sequence isunderlined and the extracellular domain is indicated in bold font.

(SEQ ID NO: 75) ATGGAGGCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCCTCGTGCTGGCGGCGGCGGCGGCGGCGG CG GCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTAC TTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATG TGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGT CTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGGCCCTTT TTCAGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGCTGGACCAGTGTGCTTC GTCTGCATCTCACTCATGTTGATGGTCTATATCTGCCACAACCGCACTGTCATTCACCATCGAGTGCCAA ATGAAGAGGACCCTTCATTAGATCGCCCTTTTATTTCAGAGGGTACTACGTTGAAAGACTTAATTTATGA TATGACAACGTCAGGTTCTGGCTCAGGTTTACCATTGCTTGTTCAGAGAACAATTGCGAGAACTATTGTG TTACAAGAAAGCATTGGCAAAGGTCGATTTGGAGAAGTTTGGAGAGGAAAGTGGCGGGGAGAAGAAGTTG CTGTTAAGATATTCTCCTCTAGAGAAGAACGTTCGTGGTTCCGTGAGGCAGAGATTTATCAAACTGTAAT GTTACGTCATGAAAACATCCTGGGATTTATAGCAGCAGACAATAAAGACAATGGTACTTGGACTCAGCTC TGGTTGGTGTCAGATTATCATGAGCATGGATCCCTTTTTGATTACTTAAACAGATACACAGTTACTGTGG AAGGAATGATAAAACTTGCTCTGTCCACGGCGAGCGGTCTTGCCCATCTTCACATGGAGATTGTTGGTAC CCAAGGAAAGCCAGCCATTGCTCATAGAGATTTGAAATCAAAGAATATCTTGGTAAAGAAGAATGGAACT TGCTGTATTGCAGACTTAGGACTGGCAGTAAGACATGATTCAGCCACAGATACCATTGATATTGCTCCAA ACCACAGAGTGGGAACAAAAAGGTACATGGCCCCTGAAGTTCTCGATGATTCCATAAATATGAAACATTT TGAATCCTTCAAACGTGCTGACATCTATGCAATGGGCTTAGTATTCTGGGAAATTGCTCGACGATGTTCC ATTGGTGGAATTCATGAAGATTACCAACTGCCTTATTATGATCTTGTACCTTCTGACCCATCAGTTGAAG AAATGAGAAAAGTTGTTTGTGAACAGAAGTTAAGGCCAAATATCCCAAACAGATGGCAGAGCTGTGAAGC CTTGAGAGTAATGGCTAAAATTATGAGAGAATGTTGGTATGCCAATGGAGCAGCTAGGCTTACAGCATTG CGGATTAAGAAAACATTATCGCAACTCAGTCAACAGGAAGGCATCAAAATG

A nucleic acid sequence encoding an processed extracellular ALK5polypeptide is as follows:

(SEQ ID NO: 76) GCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTT GTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTG TATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCT GTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGGCCCTTTTT CAGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTG

In certain embodiments, the disclosure relates to heteromultimers thatcomprise at least one ALK5 polypeptide, which includes fragments,functional variants, and modified forms thereof. Preferably, ALK5polypeptides for use in accordance with the disclosure (e.g.,heteromultimers comprising an ALK5 polypeptide and uses thereof) aresoluble (e.g., an extracellular domain of ALK5). In other preferredembodiments, ALK5 polypeptides for use in accordance with the disclosurebind to and/or inhibit (antagonize) activity (e.g., Smad signaling) ofone or more TGF-beta superfamily ligands. In some embodiments,heteromultimers of the disclosure comprise at least one ALK5 polypeptidethat is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ IDNOs: 69, 70, 73, or 74. In some embodiments, heteromultimers of thedisclosure comprise at least one ALK5 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a polypeptide that begins at any one of amino acids of25-36 (e.g., amino acid residues 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, or 36) of SEQ ID NO: 69, and ends at any one of amino acids 101-126(e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, or 126) of SEQ ID NO: 69. In some embodiments, heteromultimersof the disclosure comprise at least one ALK5 polypeptide that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to amino acids of 25-101 of SEQ ID NO: 69. Insome embodiments, heteromultimers of the disclosure comprise at leastone ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids of25-126 of SEQ ID NO: 69. In some embodiments, heteromultimers of thedisclosure comprise at least one ALK5 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids of 36-101 of SEQ ID NO: 69. In someembodiments, heteromultimers of the disclosure comprise at least oneALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids of36-126 of SEQ ID NO: 69. In some embodiments, heteromultimers of thedisclosure comprise at least one ALK5 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a polypeptide that begins at any one of amino acids of25-36 (e.g., amino acid residues 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, or 36) of SEQ ID NO: 73, and ends at any one of amino acids 101-130(e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129 or 130) of SEQ ID NO: 73. In someembodiments, heteromultimers of the disclosure comprise at least oneALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids of25-101 of SEQ ID NO: 73. In some embodiments, heteromultimers of thedisclosure comprise at least one ALK5 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids of 25-130 of SEQ ID NO: 73. In someembodiments, heteromultimers of the disclosure comprise at least oneALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids of36-101 of SEQ ID NO: 73. In some embodiments, heteromultimers of thedisclosure comprise at least one ALK5 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids of 36-130 of SEQ ID NO: 73.

As described above, the disclosure provides TβRII or betaglycanpolypeptides sharing a specified degree of sequence identity orsimilarity to a naturally occurring TβRII or betaglycan polypeptide. Todetermine the percent identity of two amino acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The amino acid residues atcorresponding amino acid positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid “identity” isequivalent to amino acid “homology”). The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

In one embodiment, the percent identity between two amino acid sequencesis determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package (available at http://www.gcg.com). In aspecific embodiment, the following parameters are used in the GAPprogram: either a Blosum 62 matrix or a PAM250 matrix, and a gap weightof 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or6. In yet another embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1):387(1984)) (available at http://www.gcg.com). Exemplary parameters includeusing a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified,percent identity between two amino acid sequences is to be determinedusing the GAP program using a Blosum 62 matrix, a GAP weight of 10 and alength weight of 3, and if such algorithm cannot compute the desiredpercent identity, a suitable alternative disclosed herein should beselected.

In another embodiment, the percent identity between two amino acidsequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

Another embodiment for determining the best overall alignment betweentwo amino acid sequences can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.,6:237-245 (1990)). In a sequence alignment the query and subjectsequences are both amino acid sequences. The result of said globalsequence alignment is presented in terms of percent identity. In oneembodiment, amino acid sequence identity is performed using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci., 6:237-245 (1990)). In a specific embodiment, parametersemployed to calculate percent identity and similarity of an amino acidalignment comprise: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5 and Gap Size Penalty=0.05.

Polypeptides of the disclosure (e.g., TβRII or betaglycan polypeptides)may additionally include any of various leader sequences at theN-terminus. Such a sequence would allow the peptides to be expressed andtargeted to the secretion pathway in a eukaryotic system. See, e.g.,Ernst et al., U.S. Pat. No. 5,082,783 (1992). Alternatively, a nativesignal sequence (e.g., native TβRII or betaglycan signal sequence) maybe used to effect extrusion from the cell. Possible leader sequencesinclude native leaders, tissue plasminogen activator (TPA) and honeybeemellitin (SEQ ID NOs. 22-24, respectively). Examples of TβRII-Fc fusionproteins incorporating a TPA leader sequence include SEQ ID NOs: 11, 13,15 and 17. Processing of signal peptides may vary depending on theleader sequence chosen, the cell type used and culture conditions, amongother variables, and therefore actual N-terminal start sites for maturepolypeptides may shift by 1, 2, 3, 4 or 5 amino acids in either theN-terminal or C-terminal direction. Examples of TβRII-Fc fusion proteinsinclude SEQ ID NOs: 11, 13, 15 and 17. It will be understood by one ofskill in the art that corresponding variants based on the long isoformof TβRII will include the 25-amino acid insertion along with aconservative Val-Ile substitution at the flanking position C-terminal tothe insertion.

In some embodiments, any of the TβRII polypeptides disclosed herein areat least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to theamino acid sequence of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36,37, 38, 39, or 48, but lack one or more N-terminal amino acids ascompared to the amino acid sequences of SEQ ID NO: 18, 20, 27, 30, 34,36, 37, 38, 39, or 48. In some embodiments, the TβRII polypeptide lacksthe amino acid corresponding to the first amino acid (threonine) of anyone of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39, or 48. In someembodiments, the TβRII polypeptide lacks the amino acids correspondingto the first and second amino acids (threonine and isoleucine,respectively) of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38,39, or 48. In some embodiments, the TβRII polypeptide lacks the aminoacids corresponding to the first, second and third amino acids(threonine, isoleucine, and proline, respectively) of any one of SEQ IDNOs: 18, 20, 27, 30, 34, 36, 37, 38, 39, or 48. In some embodiments, theTβRII polypeptide lacks the amino acids corresponding to the first,second, third and fourth amino acids (threonine, isoleucine, proline,proline, respectively) of any one of SEQ ID NOs: 18, 20, 27, 30, 34, 36,37, 38, 39, or 48.

In some embodiments, any of the TβRII polypeptides disclosed herein areat least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to theamino acid sequence of any one of SEQ ID NOs: 18 or 48, but lack theamino acid corresponding to the first amino acid (threonine) of SEQ IDNO: 18 or 48. In some embodiments, the TβRII polypeptide lacks the aminoacids corresponding to the first and second amino acids (threonine andisoleucine, respectively) of SEQ ID NO: 18 or 48. In some embodiments,the TβRII polypeptide lacks the amino acids corresponding to the first,second and third amino acids (threonine, isoleucine, and proline,respectively) of SEQ ID NO: 18 or 48. In some embodiments, the TβRIIpolypeptide lacks the amino acids corresponding to the first, second,third and fourth amino acids (threonine, isoleucine, proline, proline,respectively) of SEQ ID NO: 18 or 48.

In some embodiments, the disclosure provides for a compositioncomprising a mixture of TβRII polypeptides, wherein the TβRIIpolypeptides in the composition each comprise an amino acid sequencethat is at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100%identical to the amino acid sequence of any one of SEQ ID NOs: 18, 20,27, 30, 34, 36, 37, 38, 39, or 48; but wherein at least a portion of theTβRII polypeptides (e.g., at least 1%, 3%, 4%, 5%0, 1, 15%, 20%, 25%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) in the composition include theamino acids corresponding to the first, second, third and fourth aminoacids (threonine, isoleucine, proline and proline, respectively) of anyone of SEQ ID NOs: 18, 20, 27, 30, 34, 36, 37, 38, 39, or 48; andwherein at least a portion of the TβRII polypeptides (e.g., at least 1%,3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%)in the composition lack one or more of the amino acids corresponding tothe first, second, third and fourth amino acids (threonine, isoleucine,proline and proline, respectively) of any one of SEQ ID NOs: 18, 20, 27,30, 34, 36, 37, 38, 39, or 48. In some embodiments, the disclosureprovides for a composition comprising a mixture of TβRII polypeptides,wherein the TβRII polypeptides are at least 80%, 85%, 90%, 92%, 94%,95%, 97%, 99% or 100% identical to the amino acid sequence of any one ofSEQ ID NOs: 18 or 48, but wherein at least 30% to 80% of the TβRIIpolypeptides in the composition lack the amino acid corresponding to thefirst amino acid (threonine) of SEQ ID NO: 18 or 48. In someembodiments, the TβRII polypeptides are at least 80%, 85%, 90%, 92%,94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of anyone of SEQ ID NOs: 63-68. In some embodiments, the TβRII polypeptidesare at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 68.

In certain embodiments, the present disclosure contemplates specificmutations of the polypeptides (e.g., TβRII or betaglycan polypeptides)so as to alter the glycosylation of the polypeptide. Such mutations maybe selected so as to introduce or eliminate one or more glycosylationsites, such as O-linked or N-linked glycosylation sites.Asparagine-linked glycosylation recognition sites generally comprise atripeptide sequence, asparagine-X-threonine (or asparagine-X-serine)(where “X” is any amino acid) which is specifically recognized byappropriate cellular glycosylation enzymes. The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to the sequence of the wild-type polypeptide (forO-linked glycosylation sites). A variety of amino acid substitutions ordeletions at one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 published Sep. 11, 1987, and inAplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306,incorporated by reference herein. Removal of one or more carbohydratemoieties present on a polypeptide may be accomplished chemically and/orenzymatically. Chemical deglycosylation may involve, for example,exposure of the polypeptide to the compound trifluoromethanesulfonicacid, or an equivalent compound. This treatment results in the cleavageof most or all sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.Chemical deglycosylation is further described by Hakimuddin et al.(1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal.Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol.138:350. The sequence of a polypeptide may be adjusted, as appropriate,depending on the type of expression system used, as mammalian, yeast,insect and plant cells may all introduce differing glycosylationpatterns that can be affected by the amino acid sequence of the peptide.In general, polypeptides (e.g., TβRII or betaglycan polypeptides) foruse in humans will be expressed in a mammalian cell line that providesproper glycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines, yeast cell lines with engineeredglycosylation enzymes, and insect cells are expected to be useful aswell.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of a polypeptide (e.g., TβRIIor betaglycan polypeptides), as well as truncation mutants; pools ofcombinatorial mutants are especially useful for identifying functionalvariant sequences. The purpose of screening such combinatorial librariesmay be to generate, for example, polypeptide variants which can act aseither agonists or antagonist, or alternatively, which possess novelactivities all together. A variety of screening assays are providedbelow, and such assays may be used to evaluate variants. For example, aTβRII polypeptide variant may be screened for ability to bind to a TβRIIligand, to prevent binding of a TβRII ligand to a TβRII polypeptide orto interfere with signaling caused by a TβRII ligand. The activity of aTβRII polypeptide or its variants may also be tested in a cell-based orin vivo assay, particularly any of the assays disclosed in the Examples.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to a polypeptide (e.g., TβRII orbetaglycan polypeptides) comprising an extracellular domain of anaturally occurring polypeptide. Likewise, mutagenesis can give rise tovariants which have serum half-lives dramatically different than thecorresponding wild-type polypeptide. For example, the altered proteincan be rendered either more stable or less stable to proteolyticdegradation or other processes which result in destruction of, orotherwise elimination or inactivation of, a native TβRII polypeptide.Such variants, and the genes which encode them, can be utilized to alterTβRII polypeptide levels by modulating the half-life of the TβRIIpolypeptides. For instance, a short half-life can give rise to moretransient biological effects and can allow tighter control ofrecombinant polypeptide levels within the patient. In an Fc fusionprotein, mutations may be made in the linker (if any) and/or the Fcportion to alter the half-life of the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential polypeptide (e.g., TβRII or betaglycanpolypeptides) sequences. For instance, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential polypeptide nucleotide sequencesare expressible as individual polypeptides, or alternatively, as a setof larger fusion proteins (e.g., for phage display).

There are many ways by which the library of potential polypeptide (e.g.,TβRII or betaglycan polypeptide) variants can be generated from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be carried out in an automatic DNA synthesizer, andthe synthetic genes then be ligated into an appropriate vector forexpression. The synthesis of degenerate oligonucleotides is well knownin the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakuraet al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos.Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakuraet al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984)Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). Suchtechniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al., (1990) Science 249:386-390;Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990)Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, polypeptide (e.g., TβRII orbetaglycan polypeptide) variants can be generated and isolated from alibrary by screening using, for example, alanine scanning mutagenesisand the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al.,(1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601;Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al.,(1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989)Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al.,(1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol.12:2644-2652; McKnight et al., (1982) Science 232:316); by saturationmutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis(Leung et al., (1989) Method Cell Mol Biol 1:11-19); or by randommutagenesis, including chemical mutagenesis, etc. (Miller et al., (1992)A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor,N.Y.; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linkerscanning mutagenesis, particularly in a combinatorial setting, is anattractive method for identifying truncated (bioactive) forms ofpolypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of polypeptides (e.g., TβRII or betaglycanpolypeptides). The most widely used techniques for screening large genelibraries typically comprises cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. Preferred assays include ligand binding assays andligand-mediated cell signaling assays.

In certain embodiments, the polypeptides (e.g., TβRII or betaglycanpolypeptides) of the disclosure may further comprise post-translationalmodifications in addition to any that are naturally present in thenative polypeptides. Such modifications include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,pegylation (polyethylene glycol) and acylation. As a result, themodified polypeptides may contain non-amino acid elements, such aspolyethylene glycols, lipids, mono- or poly-saccharides, and phosphates.Effects of such non-amino acid elements on the functionality of apolypeptide may be tested as described herein for other polypeptidevariants. When a polypeptide is produced in cells by cleaving a nascentform of the polypeptide, post-translational processing may also beimportant for correct folding and/or function of the protein. Differentcells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK-293) havespecific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the polypeptides.

In certain aspects, the disclosure provides for fusion proteins (e.g.,TβRII or betaglycan fusion proteins), and in some embodiments, a firstportion (e.g., a TβRII or betaglycan polypeptide portion) is connectedto a heterologous portion (e.g., Fc portion) by means of a linker. Insome embodiments, the linkers are glycine and serine rich linkers. Othernear neutral amino acids, such as, but not limited to, Thr, Asn, Pro andAla, may also be used in the linker sequence. In some embodiments, thelinker comprises various permutations of amino acid sequences containingGly and Ser. In some embodiments, the linker is greater than 10 aminoacids in length. In further embodiments, the linkers have a length of atleast 12, 15, 20, 21, 25, 30, 35, 40, 45 or 50 amino acids. In someembodiments, the linker is less than 40, 35, 30, 25, 22 or 20 aminoacids. In some embodiments, the linker is 10-50, 10-40, 10-30, 10-25,10-21, 10-15, 10, 15-25, 17-22, 20, or 21 amino acids in length. In somepreferred embodiments, the linker comprises the amino acid sequenceGlyGlyGlyGlySer (GGGGS) (SEQ ID NO: 19), or repetitions thereof(GGGGS)n, where n≥2. In particular embodiments n≥3, or n=3-10. Theapplication teaches the surprising finding that proteins comprising aTβRII portion and a heterologous portion fused together by means of a(GGGGS)₄ linker were associated with a stronger affinity for TGFβ1 andTGFβ3 as compared to a TβRII fusion protein where n<4. As such, inpreferred embodiments, n≥4, or n=4-10. The application also teaches thatproteins comprising (GGGGS)n linkers in which n>4 had similar inhibitoryproperties as proteins having the (GGGGS)₄ linker. As such, in someembodiments, n is not greater than 4 in a (GGGGS)n linker. In someembodiments, n=4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, or 5-6. In someembodiments, n=3, 4, 5, 6, or 7. In particular embodiments, n=4. In someembodiments, a linker comprising a (GGGGS)_(n) sequence also comprisesan N-terminal threonine. In some embodiments, the linker is any one ofthe following:

(SEQ ID NO: 21) GGGGSGGGGS (SEQ ID NO: 4) TGGGGSGGGGS (SEQ ID NO: 5)TGGGGSGGGGSGGGGS (SEQ ID NO: 6) TGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 25)TGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 26)TGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS or (SEQ ID NO: 40)TGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.In some embodiments, the linker comprises the amino acid sequence ofTGGGPKSCDK (SEQ ID NO: 7). In some embodiments, the linker is any one ofSEQ ID NOs: 21, 4-7, 25-26 or 40 lacking the N-terminal threonine. Insome embodiments, the linker does not comprise the amino acid sequenceof SEQ ID NO: 26 or 40.

In certain aspects, functional variants or modified forms of the TβRII(or betaglycan) polypeptides include fusion proteins having at least aportion of the TβRII (or betaglycan) polypeptides and one or moreheterologous portions. Well-known examples of such heterologous portionsinclude, but are not limited to, polyhistidine, Glu-Glu, glutathione Stransferase (GST), thioredoxin, protein A, protein G, an immunoglobulinheavy chain constant region (Fc), maltose binding protein (MBP), orhuman serum albumin. A heterologous portion may be selected so as toconfer a desired property. For example, some heterologous portions areparticularly useful for isolation of the fusion proteins by affinitychromatography. For the purpose of affinity purification, relevantmatrices for affinity chromatography, such as glutathione-, amylase-,and nickel- or cobalt-conjugated resins are used. Many of such matricesare available in “kit” form, such as the Pharmacia GST purificationsystem and the QIAexpress™ system (Qiagen) useful with (HIS6) fusionpartners. As another example, a heterologous portion may be selected soas to facilitate detection of the TβRII (or betaglycan) polypeptides.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the heterologous portions have a protease cleavagesite, such as for Factor Xa or Thrombin, which allows the relevantprotease to partially digest the fusion proteins and thereby liberatethe recombinant proteins therefrom. The liberated proteins can then beisolated from the heterologous portion by subsequent chromatographicseparation. In certain preferred embodiments, a TβRII (or betaglycan)polypeptide is fused with a domain that stabilizes the TβRII polypeptidein vivo (a “stabilizer” domain). By “stabilizing” is meant anything thatincreases serum half life, regardless of whether this is because ofdecreased destruction, decreased clearance by the kidney, or otherpharmacokinetic effect. Fusions with the Fc portion of an immunoglobulinare known to confer desirable pharmacokinetic properties on a wide rangeof proteins. Likewise, fusions to human serum albumin can conferdesirable properties. Other types of heterologous portions that may beselected include multimerizing (e.g., dimerizing, tetramerizing) domainsand functional domains.

As specific examples, the present disclosure provides fusion proteinscomprising variants of TβRII polypeptides fused to an Fc domain sequenceof SEQ ID NO: 49. Optionally, the Fc domain has one or more mutations atresidues such as Asp-265, Lys-322, and Asn-434 (numbered in accordancewith the corresponding full-length IgG). In certain cases, the mutant Fcdomain having one or more of these mutations (e.g., Asp-265 mutation)has reduced ability of binding to the Fcγ receptor relative to awildtype Fc domain. In other cases, the mutant Fc domain having one ormore of these mutations (e.g., Asn-434 mutation) has increased abilityof binding to the MHC class I-related Fc-receptor (FcRN) relative to awildtype Fc domain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, a TβRII (or betaglycan) polypeptide may beplaced C-terminal to a heterologous domain, or, alternatively, aheterologous domain may be placed C-terminal to a TβRII (or betaglycan)polypeptide. The TβRII (or betaglycan) polypeptide domain and theheterologous domain need not be adjacent in a fusion protein, andadditional domains or amino acid sequences may be included C- orN-terminal to either domain or between the domains.

As used herein, the term “immunoglobulin Fc domain” or simply “Fc” isunderstood to mean the carboxyl-terminal portion of an immunoglobulinchain constant region, preferably an immunoglobulin heavy chain constantregion, or a portion thereof. For example, an immunoglobulin Fc regionmay comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2domain and a CH3 domain, or 5) a combination of two or more domains andan immunoglobulin hinge region. In a preferred embodiment theimmunoglobulin Fc region comprises at least an immunoglobulin hingeregion a CH2 domain and a CH3 domain, and preferably lacks the CH1domain. In some embodiments, the immunoglobulin Fc region is a humanimmunoglobulin Fc region.

In one embodiment, the class of immunoglobulin from which the heavychain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or4).

An example of a native amino acid sequence that may be used for the Fcportion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 58). Dottedunderline indicates the hinge region, and solid underline indicatespositions with naturally occurring variants. In part, the disclosureprovides polypeptides comprising, consisting essential of, or consistingof amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity toSEQ ID NO: 58. Naturally occurring variants in G1Fe would include E134Dand M136L according to the numbering system used in SEQ ID NO: 58 (seeUniprot P01857).

(SEQ ID NO: 58)

 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV201 FSCSVMHEAL HNHYTQKSLS LSPGK

Optionally, the IgG1 Fe domain has one or more mutations at residuessuch as Asp-265, lysine 322, and Asn-434. In certain cases, the mutantIgG1 Fe domain having one or more of these mutations (e.g., Asp-265mutation) has reduced ability of binding to the Fcγ receptor relative toa wild-type Fe domain. In other cases, the mutant Fe domain having oneor more of these mutations (e.g., Asn-434 mutation) has increasedability of binding to the MHC class I-related Fc-receptor (FcRN)relative to a wild-type IgG1 Fe domain.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 59). Dottedunderline indicates the hinge region and double underline indicatespositions where there are data base conflicts in the sequence (accordingto UniProt P01859). In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 59.

(SEQ ID NO: 59)

 51 FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS101 NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP151 SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS201 CSVMHEALHN HYTQKSLSLS PGK

Two examples of amino acid sequences that may be used for the Fc portionof human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be upto four times as long as in other Fc chains and contains three identical15-residue segments preceded by a similar 17-residue segment. The firstG3Fc sequence shown below (SEQ ID NO: 60) contains a short hinge regionconsisting of a single 15-residue segment, whereas the second G3Fcsequence (SEQ ID NO: 61) contains a full-length hinge region. In eachcase, dotted underline indicates the hinge region, and solid underlineindicates positions with naturally occurring variants according toUniProt P01859. In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 60 or61.

(SEQ ID NO: 60)

 51 VSHEDPEVQF KWYVDGVEVH NAKTKPREEQ YNSTFRVVSV LTVLHQDWLN101 GKEYKCKVSN KALPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL151 TCLVKGFYPS DIAVEWESSG QPENNYNTTP PMLDSDGSFF LYSKLTVDKS201 RWQQGNIFSC SVMHEALHNR FTQKSLSLSP GK (SEQ ID NO: 61)

101 EDPEVQFKWY VDGVEVHNAK TKPREEQYNS TFRVVSVLTV LHQDWLNGKE151 YKCKVSNKAL PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL201 VKGFYPSDIA VEWESSGQPE NNYNTTPPML DSDGSFFLYS KLTVDKSRWQ251 QGNIFSCSVM HEALHNRFTQ KSLSLSPGK

Naturally occurring variants in G3Fc (for example, see Uniprot P01860)include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del,F221Y when converted to the numbering system used in SEQ ID NO: 60, andthe present disclosure provides fusion proteins comprising G3Fc domainscontaining one or more of these variations. In addition, the humanimmunoglobulin IgG3 gene (IGHG3) shows a structural polymorphismcharacterized by different hinge lengths [see Uniprot P01859].Specifically, variant WIS is lacking most of the V region and all of theCH1 region. It has an extra interchain disulfide bond at position 7 inaddition to the 11 normally present in the hinge region. Variant ZUClacks most of the V region, all of the CH1 region, and part of thehinge. Variant OMM may represent an allelic form or another gamma chainsubclass. The present disclosure provides additional fusion proteinscomprising G3Fc domains containing one or more of these variants.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 62). Dottedunderline indicates the hinge region. In part, the disclosure providespolypeptides comprising, consisting essential of, or consisting of aminoacid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:62.

(SEQ ID NO: 62)

 51 EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE101 YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL151 VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ201 EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

A variety of engineered mutations in the Fc domain are presented hereinwith respect to the G1Fc sequence (SEQ ID NO: 58), and analogousmutations in G2Fc, G3Fc, and G4Fc can be derived from their alignmentwith G1Fc in FIG. 7 Due to unequal hinge lengths, analogous Fc positionsbased on isotype alignment (FIG. 7) possess different amino acid numbersin SEQ ID NOs: 58, 59, 60, 61, and 62. It can also be appreciated that agiven amino acid position in an immunoglobulin sequence consisting ofhinge, C_(H)2, and C_(H)3 regions (e.g., SEQ ID NOs: 58, 59, 60, 61, and62) will be identified by a different number than the same position whennumbering encompasses the entire IgG1 heavy-chain constant domain(consisting of the C_(H)1, hinge, C_(H)2, and C_(H)3 regions) as in theUniprot database.

Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igε) and IgM(Igμ), may be used. The choice of appropriate immunoglobulin heavy chainconstant region is discussed in detail in U.S. Pat. Nos. 5,541,087 and5,726,044. The choice of particular immunoglobulin heavy chain constantregion sequences from certain immunoglobulin classes and subclasses toachieve a particular result is considered to be within the level ofskill in the art. The portion of the DNA construct encoding theimmunoglobulin Fc region preferably comprises at least a portion of ahinge domain, and preferably at least a portion of a CH3 domain of Fcgamma or the homologous domains in any of IgA, IgD, IgE, or IgM.

Furthermore, it is contemplated that substitution or deletion of aminoacids within the immunoglobulin heavy chain constant regions may beuseful in the practice of the methods and compositions disclosed herein.One example would be to introduce amino acid substitutions in the upperCH2 region to create an Fc variant with reduced affinity for Fcreceptors (Cole et al. (1997) J. Immunol. 159:3613).

In some embodiments, the disclosure provides for TβRII polypeptidesfusion proteins comprising an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NOs: 11, 13,15 and 17, or biologically active fragments thereof. In someembodiments, the TβRII polypeptides fusion proteins comprise an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofany one of SEQ ID NOs: 11, 13, and 15, or biologically active fragmentsthereof. In some embodiments, the TβRII polypeptides fusion proteinscomprise an amino acid sequence that is at least 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence of any one of SEQ ID NO: 13, or a biologicallyactive fragment thereof. In some embodiments, the TβRII polypeptidesfusion proteins comprise an amino acid sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of any one of SEQ ID NO: 63, or a biologically active fragmentthereof. In some embodiments, the TβRII polypeptides fusion proteinscomprise an amino acid sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of any one ofSEQ ID NO: 48, or a biologically active fragment thereof. In someembodiments, the TβRII polypeptides fusion proteins comprise an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NO: 64, or abiologically active fragment thereof. In some embodiments, the TβRIIpolypeptides fusion proteins comprise an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence of any one of SEQ ID NO: 65, or a biologically activefragment thereof. In some embodiments, the TβRII polypeptides fusionproteins comprise an amino acid sequence that is at least 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of anyone of SEQ ID NO: 66, or a biologically active fragment thereof. In someembodiments, the TβRII polypeptides fusion proteins comprise an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NO: 67, or abiologically active fragment thereof. In some embodiments, the TβRIIpolypeptides fusion proteins comprise an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence of any one of SEQ ID NO: 68, or a biologically activefragment thereof. In some embodiments, the TβRII polypeptides fusionprotein comprises an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe amino acid sequence of SEQ ID NO: 49, or a biologically activefragment thereof.

In some embodiments, the fusion proteins described herein have improvedbinding affinity for TGFβ1 and TGFβ3. In some embodiments, a fusionprotein comprising a linker at least 10 amino acids in length (e.g., afusion protein having the amino acid sequence of any one of SEQ ID NOs:11, 13, 15, 48, and 63-68) has improved binding affinity for TGFβ1 andTGFβ3 as compared to a reference fusion protein (e.g., a fusion proteinhaving the amino acid sequence of SEQ ID NO: 9). In some embodiments,the fusion protein binds to TGFβ1 with a K_(D) of less than 200 pM, lessthan 150 pM, less than 100 pM, less than 75 pM, less than 50 pM or lessthan 25 pM. In some embodiments, the fusion protein binds to TGFβ3 witha K_(D) of less than 75 pM, less than 70 pM, less than 60 pM, less than50 pM, less than 40 pM, less than 35 pM, less than 25 pM, less than 15,less than 10, or less than 5 pM.

In some embodiments any of the polypeptides disclosed herein inhibitsTGFβ1 and/or TGFβ3 in a measurable assay. In some embodiments, thepolypeptide inhibits TGFβ1 with an IC₅₀ of less than 1.0, 0.9, 0.8, 0.7,0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.08, 0.09, 0.07, 0.06, 0.05, 0.04, 0.03,or 0.02 nM, as determined using a reporter gene assay. In someembodiments, the polypeptide inhibits TGFβ3 with an IC₅₀ of less than1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07,0.06, 0.05, 0.04, 0.03, or 0.02 nM, as determined using a reporter geneassay. In some embodiments, the reporter gene assay is a CAGA reporterassay. In some embodiments, the CAGA assay is based on a human lungcarcinoma cell line transfected with a pGL3(CAGA)12 reporter plasmid(Dennler et al, 1998, EMBO 17: 3091-3100) as well as a Renilla reporterplasmid (pRLCMV) to control for transfection efficiency. The CAGA motifis present in the promoters of TGFβ-responsive genes (for example,PAI-1), so this vector is of general use for factors signaling throughSMAD2 and SMAD3. See, e.g., Example 2.

In some embodiments, the disclosure provides for TβRII (or betaglycan)containing fusion polypeptides. The fusion polypeptides may be preparedaccording to any of the methods disclosed herein or that are known inthe art.

In some embodiments, any of the fusion polypeptides disclosed hereincomprises the following components: a) any of the TβRII (or betaglycan)polypeptides disclosed herein (“A”), b) any of the linkers disclosedherein (“B”), c) any of the heterologous portions disclosed herein(“C”), and optionally a linker (“X”). In such embodiments, the fusionpolypeptide may be arranged in a manner as follows (N-terminus toC-terminus): A-B-C or C-B-A. In such embodiments, the fusion polypeptidemay be arranged in a manner as follows (N-terminus to C-terminus):X-A-B-C or X-C-B-A. In some embodiments, the fusion polypeptidecomprises each of A, B and C (and optionally a leader sequence such asthe amino acid sequence of SEQ ID NO: 23), and comprises no more than100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 additionalamino acids (but which may include further post-translationalmodifications, such as PEGylation).

In some embodiments, the fusion polypeptide comprises a leader sequence(e.g., SEQ ID NO: 23) positioned in a manner as follows (N-terminus toC-terminus): X-A-B-C, and the fusion polypeptide comprises 1, 2, 3, 4,or 5 amino acids between X and A. In some embodiments, the fusionpolypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positionedin a manner as follows (N-terminus to C-terminus): X-C-B-A, and thefusion polypeptide comprises 1, 2, 3, 4, or 5 amino acids between X andC. In some embodiments, the fusion polypeptide comprises a leadersequence (e.g., SEQ ID NO: 23) positioned in a manner as follows(N-terminus to C-terminus): X-A-B-C, and the fusion polypeptidecomprises an alanine between X and A. In some embodiments, the fusionpolypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positionedin a manner as follows (N-terminus to C-terminus): X-C-B-A, and thefusion polypeptide comprises an alanine between X and C. In someembodiments, the fusion polypeptide comprises a leader sequence (e.g.,SEQ ID NO: 23) positioned in a manner as follows (N-terminus toC-terminus): X-A-B-C, and the fusion polypeptide comprises a glycine andan alanine between X and A. In some embodiments, the fusion polypeptidecomprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manneras follows (N-terminus to C-terminus): X-C-B-A, and the fusionpolypeptide comprises a glycine and an alanine between X and C. In someembodiments, the fusion polypeptide comprises a leader sequence (e.g.,SEQ ID NO: 23) positioned in a manner as follows (N-terminus toC-terminus): X-A-B-C, and the fusion polypeptide comprises a threoninebetween X and A. In some embodiments, the fusion polypeptide comprises aleader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows(N-terminus to C-terminus): X-C-B-A, and the fusion polypeptidecomprises a threonine between X and C.

In some embodiments, the fusion polypeptide comprises an amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any of the TβRII polypeptideamino acid sequences disclosed herein (e.g., SEQ ID NO: 18), wherein theTβRII polypeptide portion of the fusion polypeptide comprises no morethan 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but whichmay include further post-translational modifications, such asPEGylation). In some embodiments, the fusion polypeptide comprises anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the linkersequences disclosed herein (e.g., SEQ ID NO: 6), wherein the linkerportion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation). In some embodiments, the fusionpolypeptide comprises an amino acid sequence that is at least 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any of the heterologous portion sequences disclosed herein (e.g., SEQID NO: 49), wherein the heterologous portion of the fusion polypeptidecomprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional aminoacids (but which may include further post-translational modifications,such as PEGylation). In some embodiments, the fusion polypeptidecomprises any of the TβRII polypeptide amino acid sequences disclosedherein (e.g., SEQ ID NO: 18), wherein the TβRII polypeptide portion ofthe fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2or 1 additional amino acids (but which may include furtherpost-translational modifications, such as PEGylation). In someembodiments, the fusion polypeptide comprises any of the linkersequences disclosed herein (e.g., SEQ ID NO: 6), wherein the linkerportion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation). In some embodiments, the fusionpolypeptide comprises any of the heterologous portion sequencesdisclosed herein (e.g., SEQ ID NO: 49), wherein the heterologous portionof the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4,3, 2, or 1 additional amino acids (but which may include furtherpost-translational modifications, such as PEGylation).

In some embodiments, the disclosure provides for a fusion polypeptide,wherein the fusion polypeptide consists or consists essentially of (andnot necessarily in the following order): a) an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to any of the TβRII polypeptide amino acidsequences disclosed herein (e.g., SEQ ID NO: 18), wherein the TβRIIpolypeptide portion of the fusion polypeptide comprises no more than 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which mayinclude further post-translational modifications, such as PEGylation);b) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the linkersequences disclosed herein (e.g., SEQ ID NO: 6), wherein the linkerportion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation); and c) an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to any of the heterologous portion sequencesdisclosed herein (e.g., SEQ ID NO: 49), wherein the heterologous portionof the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4,3, 2, or 1 additional amino acids (but which may include furtherpost-translational modifications, such as PEGylation); and d) optionallya leader sequence (e.g., SEQ ID NO: 23). In some embodiments, thedisclosure provides for a fusion polypeptide, wherein the fusionpolypeptide consists or consists essentially of (and not necessarily inthe following order): a) any of the TβRII polypeptide amino acidsequences disclosed herein (e.g., SEQ ID NO: 18), wherein the TβRIIpolypeptide portion of the fusion polypeptide comprises no more than 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which mayinclude further post-translational modifications, such as PEGylation);b) any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6),wherein the linker portion of the fusion polypeptide comprises no morethan 5, 4, 3, 2 or 1 additional amino acids (but which may includefurther post-translational modifications, such as PEGylation); and c)any of the heterologous portion sequences disclosed herein (e.g., SEQ IDNO: 49), wherein the heterologous portion of the fusion polypeptidecomprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional aminoacids (but which may include further post-translational modifications,such as PEGylation); and d) optionally a leader sequence (e.g., SEQ IDNO: 23).

In some embodiments, the disclosure provides for a fusion polypeptideconsisting of or consisting essentially of (and not necessarily in thefollowing order): a) a TβRII polypeptide portion consisting of an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 18 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation); b) a linker portion consisting of anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1 additionalamino acids (but which may include further post-translationalmodifications, such as PEGylation); and c) a heterologous portionconsisting of an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe amino acid sequence of SEQ ID NO: 49 and no more than 25, 20, 15,10, 5, 4, 3, 2, or 1 additional amino acids (but which may includefurther post-translational modifications, such as PEGylation); and d)optionally a leader sequence (e.g., SEQ ID NO: 23). In some embodiments,the disclosure provides for a fusion polypeptide consisting orconsisting essentially of (and not necessarily in the following order):a) a TβRII polypeptide portion consisting of the amino acid sequence ofSEQ ID NO: 18 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation); b) a linker portion consisting ofthe amino acid sequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation); and c) a heterologous portionconsisting of the amino acid sequence of SEQ ID NO: 49 and no more than25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but which mayinclude further post-translational modifications, such as PEGylation);and d) optionally a leader sequence (e.g., SEQ ID NO: 23).

In some embodiments, the fusion protein does not comprise a leadersequence. In some embodiments, the fusion protein comprises an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 48.

(SEQ ID NO: 48) TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQK SCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDTGGGGSGGGGSGGGGSGGGGSTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, the disclosure provides for a TβRII fusionpolypeptide wherein the polypeptide does not comprise an antibody orantigen-binding portion thereof. In some embodiments, the polypeptidedoes not bind with appreciable affinity to a cytokine other than atransforming growth factor beta superfamily ligand (e.g., TGFβ1, TGFβ2and/or TGFβ3). In some embodiments, the polypeptide does not bind withappreciable affinity to a cytokine other than TGFβ1, TGFβ2 and/or TGFβ3.In some embodiments, the polypeptide does not bind with appreciableaffinity to a cytokine other than TGFβ1 and/or TGFβ3. In someembodiments, the polypeptide does not bind with appreciable affinity toCD4, CD8, CD25, CTLA-4, IL-10, TGFβ Receptor, PD-1, PD-L1, PD-L2, RANK,RANKL, HER2/neu, EGFR1, CD20, VEGF, TNF-α, TNFR2, FoxP3, CD80, CD86,IFN-α, IFN-β, IFN-γ, GITR, 4-1BB, OX-40, TLR1-10, ErbB-1, HER1,ErbB-3/HER3, ErbB-4/HER4, IGFR, IGFBP, IGF-1R, PDGFR, FGFR, VEGFR, HGFR,TRK receptor, ephrin receptors, AXL receptors, LTK receptors, TIEreceptors, angiopoietin1, 2, ROR receptor, DDR receptor, RET receptor,KLG receptor, RYK receptor, MuSK receptor, ILβR, IlαR, TNTRSF, TRAILreceptor, ARTC1, alpha-actinin-4, Bcr-abl, B-RAF, caspases,beta-catenin, fibronectin, GPNMB, GDP-L, LDLR, HLA-A2, MLA-A11, HSP70,KIAA205, MART2, MUM-1, 2, 3, PAP, neo-PAP, NFYC, OGT, OS-9, pml-RARalphafusion protein, PRDX5, PTPRK, KRAS2, NRAS, HRAS, RBAF600, SIRT2. SNRPDI,SYT-SSX1 or -SSX2 fusion protein, Triosephosphate Isomerase, BAGE,BAGE-1. BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V, HERV-K MEL,KK-LC, KM-HIN-1, LAGE, LAGE-1, CAMEL, MAGE-1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10. MAGE-A11, MAGE-A12,MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5. MAGE-B6, MAGE-C1, MAGE-C2, mucin 1(MUC1), MART-1/Melan-A (MLANA), gp100, gp100/Pme117 (SILV), tyrosinase(TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2, SAGE, Sp17. SSX-1,2, 3, 4, TRP2-1NT2, carcino-embryonic antigen (CEA), Kallikfein 4,mammaglobm-A, OA1, prostate specific antigen (PSA), prostate specificmembrane antigen, TRP-1/, 75. TRP-2, AIM-2. BING-4, CPSF, cyclin D1,Ep-CAM, EpbA3, FGF-5, gp250, iCE), AFP, M-CSF, mdm-2, MUC1, p53 (TP53),PBF, FRAME, PSMA, RAGE-1. RNF43, RU2AS, SOX10, STEAP1, survivin (BIRCS),hTERT, telomerase, WTi, SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1,CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15qI4, HCA66I, LDHC,MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE,TPTE, estrogen receptors (ER), androgen receptors (AR), CD40, CD30,CD20, CD19, CD33, CD4, CD25, CD3, CA 72-4, CA 15-3, CA 27-29, CA 125, CA19-9, beta-human chorionic gonadotropin, 1-2 microglobulin, squamouscell carcinoma antigen, neuron-specific enoJase, heat shock proteingp96, GM2, sargramostim, CTLA-4, 707-AP, ART-4, CAP-1, CLCA2, Cyp-B,HST-2, HPV proteins, EBV proteins, Hepatitis B or C virus proteins,and/or HIV proteins.

In some embodiments, the disclosure provides for a TβRII fusionpolypeptide wherein the polypeptide does not comprise an additionalligand binding domain in addition to the TβRII domain. In someembodiments, the polypeptide comprises a linear amino acid sequencecomprising a TβRII domain and a heterologous portion (e.g., an Fcportion), but the linear amino acid sequence does not comprise anyadditional ligand binding domains. In some embodiments, the polypeptidecomprises a linear amino acid sequence comprising a TβRII domain and anFc portion, but the linear amino acid sequence does not comprise anyadditional ligand binding domains. In some embodiments, the disclosureprovides for a TβRII fusion polypeptide wherein the polypeptide does notcomprise multiple ligand binding domains in a single linear amino acidsequence. In some embodiments, the disclosure provides for a TβRIIfusion polypeptide wherein the polypeptide does not comprise more thanone continuous linker sequence in a single linear amino acid sequence.In some embodiments, the polypeptide does not comprise multiplecontinuous glycine and/or serine linkers (e.g., a linker comprising(GGGGS)n, wherein n=>4) in a single linear amino acid sequence. In someembodiments, the disclosure provides for a TβRII fusion polypeptidewherein the heterologous portion is an Fc domain, and wherein only onecontinuous linker is covalently bound to the Fc domain. In someembodiments, the only one continuous linker comprises or consists of a(GGGGS)n linker, wherein n=>4.

The application further provides Fc fusion proteins with engineered orvariant Fc regions. Such antibodies and Fc fusion proteins may beuseful, for example, in modulating effector functions, such as,antigen-dependent cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC). Additionally, the modifications may improve thestability of the antibodies and Fc fusion proteins. Amino acid sequencevariants of the antibodies and Fc fusion proteins are prepared byintroducing appropriate nucleotide changes into the DNA, or by peptidesynthesis. Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequences of the antibodies and Fc fusion proteins disclosed herein. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the antibodies and Fc fusion proteins,such as changing the number or position of glycosylation sites.

Antibodies and Fc fusion proteins with reduced effector function may beproduced by introducing changes in the amino acid sequence, including,but are not limited to, the Ala-Ala mutation described by Bluestone etal. (see WO 94/28027 and WO 98/47531; also see Xu et al. 2000 CellImmunol 200; 16-26). Thus, in certain embodiments, Fc fusion proteins ofthe disclosure with mutations within the constant region including theAla-Ala mutation may be used to reduce or abolish effector function.According to these embodiments, antibodies and Fc fusion proteins maycomprise a mutation to an alanine at position 234 or a mutation to analanine at position 235, or a combination thereof. In one embodiment,the antibody or Fc fusion protein comprises an IgG4 framework, whereinthe Ala-Ala mutation would describe a mutation(s) from phenylalanine toalanine at position 234 and/or a mutation from leucine to alanine atposition 235. In another embodiment, the antibody or Fc fusion proteincomprises an IgG1 framework, wherein the Ala-Ala mutation would describea mutation(s) from leucine to alanine at position 234 and/or a mutationfrom leucine to alanine at position 235. The antibody or Fc fusionprotein may alternatively or additionally carry other mutations,including the point mutation K322A in the CH2 domain (Hezareh et al.2001 J Virol. 75: 12161-8).

In particular embodiments, the antibody or Fc fusion protein may bemodified to either enhance or inhibit complement dependent cytotoxicity(CDC). Modulated CDC activity may be achieved by introducing one or moreamino acid substitutions, insertions, or deletions in an Fc region (see,e.g., U.S. Pat. No. 6,194,551). Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved or reduced internalizationcapability and/or increased or decreased complement-mediated cellkilling. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes,B. J. Immunol. 148:2918-2922 (1992), WO99/51642, Duncan & Winter Nature322: 738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO94/29351.

B. Nucleic Acids and Methods of Manufacture

In certain embodiments, the present disclosure makes available isolatedand/or purified forms of polypeptides (e.g., TβRII or betaglycanpolypeptides), which are isolated from, or otherwise substantially freeof (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% free of), otherproteins and/or other polypeptide species. Polypeptides will generallybe produced by expression from recombinant nucleic acids.

In certain embodiments, the disclosure includes nucleic acids encodingsoluble polypeptides (e.g., TβRII or betaglycan polypeptides) comprisingthe coding sequence for an extracellular portion of a protein (e.g., aTβRII or betaglycan protein). In further embodiments, this disclosurealso pertains to a host cell comprising such nucleic acids. The hostcell may be any prokaryotic or eukaryotic cell. For example, apolypeptide of the present disclosure may be expressed in bacterialcells such as E. coli, insect cells (e.g., using a baculovirusexpression system), yeast, or mammalian cells. Other suitable host cellsare known to those skilled in the art. Accordingly, some embodiments ofthe present disclosure further pertain to methods of producing thepolypeptides.

In certain aspects, the disclosure provides isolated and/or recombinantnucleic acids encoding any of the polypeptides (e.g., TβRII orbetaglycan polypeptides), including fragments, functional variants andfusion proteins disclosed herein. SEQ ID NOs: 8, 10, 12, 14, 16, 46 or47 encode variants of TβRII extracellular domain fused to an IgG Fcdomain. The subject nucleic acids may be single-stranded or doublestranded. Such nucleic acids may be DNA or RNA molecules. These nucleicacids may be used, for example, in methods for making polypeptides or asdirect therapeutic agents (e.g., in an antisense, RNAi or gene therapyapproach).

In certain aspects, the subject nucleic acids encoding polypeptides arefurther understood to include nucleic acids that are variants of SEQ IDNOs: 8, 10, 12, 14, 16, 46 or 47. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants.

In certain embodiments, the disclosure provides isolated or recombinantnucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs:8, 10, 12, 14, 16, 46 or 47. In particular embodiments, the disclosureprovides isolated or recombinant nucleic acid sequences that are atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identical to SEQ ID NO: 12, or fragments thereof. One ofordinary skill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NOs: 8, 10, 12, 14, 16, 46 or 47, and variantsof SEQ ID NOs: 8, 10, 12, 14, 16, 46 or 47 are also within the scope ofthis disclosure. In further embodiments, the nucleic acid sequences ofthe disclosure can be isolated, recombinant, and/or fused with aheterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the disclosure also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequences designated in SEQ ID NOs: 8, 10, 12, 14, 16, 46or 47 complement sequences of SEQ ID NOs: 8, 10, 12, 14, 16, 46 or 47,or fragments thereof. As discussed above, one of ordinary skill in theart will understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. For example, one could performthe hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about45° C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In some embodiments, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 8, 10, 12, 14, 16, 46 or 47 due to degeneracy in thegenetic code are also within the scope of the disclosure. For example, anumber of amino acids are designated by more than one triplet. Codonsthat specify the same amino acid, or synonyms (for example, CAU and CACare synonyms for histidine) may result in “silent” mutations which donot affect the amino acid sequence of the protein. However, it isexpected that DNA sequence polymorphisms that do lead to changes in theamino acid sequences of the subject proteins will exist among mammaliancells. One skilled in the art will appreciate that these variations inone or more nucleotides (up to about 3-5% of the nucleotides) of thenucleic acids encoding a particular protein may exist among individualsof a given species due to natural allelic variation. Any and all suchnucleotide variations and resulting amino acid polymorphisms are withinthe scope of this disclosure.

It will be appreciated by one of skill in the art that correspondingvariants based on the long isoform of TβRII will include nucleotidesequences encoding the 25-amino acid insertion along with a conservativeVal-Ile substitution at the flanking position C-terminal to theinsertion. It will also be appreciated that corresponding variants basedon either the long (A) or short (B) isoforms of TβRII will includevariant nucleotide sequences comprising an insertion of 108 nucleotides,encoding a 36-amino-acid insertion (SEQ ID NO: 41), at the same locationdescribed for naturally occurring TβRII isoform C.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects disclosed herein, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding a polypeptide (e.g., TβRII or betaglycan polypeptide) andoperably linked to at least one regulatory sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe T polypeptide. Accordingly, the term regulatory sequence includespromoters, enhancers, and other expression control elements. Exemplaryregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences that control the expression of a DNA sequence when operativelylinked to it may be used in these vectors to express DNA sequencesencoding a polypeptide (e.g., TβRII or betaglycan polypeptide). Suchuseful expression control sequences, include, for example, the early andlate promoters of SV40, tet promoter, adenovirus or cytomegalovirusimmediate early promoter, RSV promoters, the lac system, the trp system,the TAC or TRC system, T7 promoter whose expression is directed by T7RNA polymerase, the major operator and promoter regions of phage lambda,the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other protein encoded by the vector, such as antibiotic markers,should also be considered.

A recombinant nucleic acid included in the disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant polypeptide (e.g., TβRII or betaglycan polypeptide)include plasmids and other vectors. For instance, suitable vectorsinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In certain embodiments, a vector will be designed for production of thesubject polypeptides (e.g., TβRII or betaglycan polypeptidea) in CHOcells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif), pcDN4vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega,Madison, Wis.). In a preferred embodiment, a vector will be designed forproduction of the subject polypeptides in HEK-293 cells. As will beapparent, the subject gene constructs can be used to cause expression ofthe subject polypeptides in cells propagated in culture, e.g., toproduce proteins, including fusion proteins or variant proteins, forpurification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NOs: 8, 10,12, 14, 16, 46 or 47) for one or more of the subject polypeptides (e.g.,TβRII or betaglycan polypeptides). The host cell may be any prokaryoticor eukaryotic cell. For example, a TβRII polypeptide disclosed hereinmay be expressed in bacterial cells such as E. coli, insect cells (e.g.,using a baculovirus expression system), yeast, or mammalian cells. Othersuitable host cells are known to those skilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject polypeptides (e.g., TβRII or betaglycanpolypeptides). For example, a host cell transfected with an expressionvector encoding a polypeptide can be cultured under appropriateconditions to allow expression of the polypeptide to occur. Thepolypeptide may be secreted and isolated from a mixture of cells andmedium containing the polypeptide. Alternatively, the polypeptide may beretained cytoplasmically or in a membrane fraction and the cellsharvested, lysed and the protein isolated. A cell culture includes hostcells, and media. Suitable media for cell culture are well known in theart. The subject polypeptides can be isolated from cell culture medium,host cells, or both, using techniques known in the art for purifyingproteins, including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, immunoaffinitypurification with antibodies specific for particular epitopes of thepolypeptides and affinity purification with an agent that binds to adomain fused to the polypeptide (e.g., a protein A column may be used topurify an Fc fusion). In a preferred embodiment, the polypeptide is afusion protein containing a domain which facilitates its purification.As an example, purification may be achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant polypeptide(e.g., TβRII or betaglycan polypeptide), can allow purification of theexpressed fusion protein by affinity chromatography using a Ni²⁺ metalresin. The purification leader sequence can then be subsequently removedby treatment with enterokinase to provide the purified polypeptide(e.g., see Hochuli et al., (1987) J. Chromatography 411:177; andJanknecht et al., PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

C. Antibodies

In certain aspects, a TGFβ antagonist to be used in accordance with themethods and uses disclosed herein is an antibody (TGFβ antagonistantibody) or combination of antibodies. A TGFβ antagonist antibody, orcombination of antibodies, may inhibit and/or bind to, for example, oneor more ligands (e.g., TGFβ1, TGFβ2, and TGFβ3), TβRII receptor,TβRII-associated type I receptor (e.g., ALK5), and/or TβRII co-receptor(e.g., betaglycan). In some embodiments, the ability for a TGFβantagonist antibody, or combination of antibody, to inhibit signaling(e.g., Smad signaling) and/or bind to a target is determined in an invitro or cell-based assay including, for example, those describedherein. As described herein, a TGFβ antagonist antibody, or combinationof antagonist antibodies, may be used alone or in combination with oneor more additional supportive therapies or active agents to treatheterotopic ossification, preferably preventing or reducing the severityor duration of one or more complications of heterotopic ossification

In certain embodiments, a TGFβ antagonist antibody, or combination ofantibodies, is an antibody that inhibits at least TGFβ1. Therefore, insome embodiments, a TGFβ antagonist antibody, or combination ofantibodies, binds to at least TGFβ1. As used herein, a TGFβ1 antibody(anti-TGFβ1 antibody) generally refers to an antibody that is capable ofbinding to TGFβ1 with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting TGFβ1. Incertain embodiments, the extent of binding of an anti-TGFβ1 antibody toan unrelated, non-TGFβ1 protein is less than about 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to TGFβ1as measured, for example, by a radioimmunoassay (RIA). In certainembodiments, an anti-TGFβ1 antibody binds to an epitope of TGFβ1 that isconserved among TGFβ1 from different species. In certain preferredembodiments, an anti-TGFβ1 antibody binds to human TGFβ1. In someembodiments, a TGFβ1 antibody may inhibit TGFβ1 from binding to a typeI, type II, and/or co-receptor (e.g., TβRII, ALK5, and/or betaglycan)and thus inhibit TGFβ1 signaling (e.g., Smad signaling). It should benoted that TGFβ1 shares some sequence homology to TGFβ2 and TGFβ3.Therefore antibodies that bind TGFβ1, in some embodiments, may also bindto TGFβ2 and/or TGFβ3. In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to TGFβ1 and further binds to, for example, one or moreadditional ligands (e.g., TGFβ2, TGFβ3, or TGFβ2 and TGFβ3), one or moretype I and/or type II receptors (e.g., TβRII and ALK5), and/or one ormore co-receptors (e.g., betaglycan). In some embodiments, amultispecific antibody that binds to TGFβ1 further binds to TGFβ3 butdoes not bind or does not substantially bind to TGFβ2 (e.g., binds toTGFβ2 with a K_(D) of greater than 1×10⁻⁷ M or has relatively modestbinding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments,the disclosure relates to combinations of antibodies, and uses thereof,wherein the combination of antibodies comprises a TGFβ1 antibody and oneor more additional antibodies that bind to, for example, one or moreadditional ligands (e.g., TGFβ2, TGFβ3, or TGFβ2 and TGFβ3), one or moretype I and/or type II receptors (e.g., TβRII and ALK5), and/or one ormore co-receptors (e.g., betaglycan). In some embodiments, a combinationof antibodies that comprises a TGFβ1 antibody further comprises a TGFβ3antibody but does not comprise a TGFβ2 antibody.

In certain embodiments, a TGFβ antagonist antibody, or combination ofantibodies, is an antibody that inhibits at least TGFβ2. Therefore, insome embodiments, a Tβ TGFβ RII antagonist antibody, or combination ofantibodies, binds to at least TGFβ2. As used herein, a TGFβ2 antibody(anti-TGFβ2 antibody) generally refers to an antibody that is capable ofbinding to TGFβ2 with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting TGFβ2. Incertain embodiments, the extent of binding of an anti-TGFβ2 antibody toan unrelated, non-TGFβ2 protein is less than about 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to TGFβ2as measured, for example, by a radioimmunoassay (RIA). In certainembodiments, an anti-TGFβ2 antibody binds to an epitope of TGFβ2 that isconserved among TGFβ2 from different species. In certain preferredembodiments, an anti-TGFβ2 antibody binds to human TGFβ2. In someembodiments, a TGFβ2 antibody may inhibit TGFβ2 from binding to a typeI, type II, and/or co-receptor (e.g., TβRII, ALK5, and/or betaglycan)and thus inhibit TGFβ2 signaling (e.g., Smad signaling). It should benoted that TGFβ2 shares some sequence homology to TGFβ1 and TGFβ3.Therefore antibodies that bind TGFβ2, in some embodiments, may also bindto TGFβ1 and/or TGFβ3. In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to TGFβ2 and further binds to, for example, one or moreadditional ligands (e.g., TGFβ1, TGFβ3, or TGFβ1 and TGFβ3), one or moretype I and/or type II receptors (e.g., TβRII and ALK5), and/or one ormore co-receptors (e.g., betaglycan) In some embodiments, the disclosurerelates to combinations of antibodies, and uses thereof, wherein thecombination of antibodies comprises a TGFβ2 antibody and one or moreadditional antibodies that bind to, for example, one or more additionalligands (e.g., TGFβ1, TGFβ3, or TGFβ1 and TGFβ3), one or more type Iand/or type II receptors (e.g., TβRII and ALK5), and/or one or moreco-receptors (e.g., betaglycan).

In certain embodiments, a TGFβ antagonist antibody, or combination ofantibodies, is an antibody that inhibits at least TGFβ3. Therefore, insome embodiments, a TGFβ antagonist antibody, or combination ofantibodies, binds to at least TGFβ3. As used herein, a TGFβ3 antibody(anti-TGFβ3 antibody) generally refers to an antibody that is capable ofbinding to TGFβ3 with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting TGFβ3. Incertain embodiments, the extent of binding of an anti-TGFβ3 antibody toan unrelated, non-TGFβ3 protein is less than about 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to TGFβ3as measured, for example, by a radioimmunoassay (RIA). In certainembodiments, an anti-TGFβ3 antibody binds to an epitope of TGFβ3 that isconserved among TGFβ3 from different species. In certain preferredembodiments, an anti-TGFβ3 antibody binds to human TGFβ3. In someembodiments, a TGFβ3 antibody may inhibit TGFβ3 from binding to a typeI, type II, and/or co-receptor (e.g., TβRII, ALK5, and/or betaglycan)and thus inhibit TGFβ3 signaling (e.g., Smad signaling). It should benoted that TGFβ3 shares some sequence homology to TGFβ2 and TGFβ1.Therefore antibodies that bind TGFβ3, in some embodiments, may also bindto TGFβ2 and/or TGFβ1. In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to TGFβ3 and further binds to, for example, one or moreadditional TβRII ligands (e.g., TGFβ2, TGFβ1, or TGFβ2 and TGFβ1), oneor more type I and/or type II receptors (e.g., TβRII and ALK5), and/orone or more co-receptors (e.g., betaglycan). In some embodiments, amultispecific antibody that binds to TGFβ3 does not bind or does notsubstantially bind to TGFβ2 (e.g., binds to TGFβ2 with a K_(D) ofgreater than 1×10⁻⁷ M or has relatively modest binding, e.g., about1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, a multispecificantibody that binds to TGFβ3 further binds to TGFβ1 but does not bind ordoes not substantially bind to TGFβ2 (e.g., binds to TGFβ2 with a K_(D)of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, the disclosure relatesto combinations of antibodies, and uses thereof, wherein the combinationof antibodies comprises a TGFβ3 antibody and one or more additionalantibodies that bind to, for example, one or more additional ligands(e.g., TGFβ2, TGFβ1, or TGFβ2 and TGFβ1), one or more type I and/or typeII receptors (e.g., TβRII and ALK5), and/or one or more co-receptors(e.g., betaglycan). In some embodiments, a combination of antibodiesthat comprises a TGFβ3 antibody further comprises a TGFβ1 antibody butdoes not comprise a TGFβ2 antibody.

In some embodiments, the TGFβ antagonist antibody binds to all threeTGFβ isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments theTGFβ antagonist antibody is fresolimumab.

In certain aspects, a TGFβ antagonist antibody, or combination ofantibodies, is an antibody that inhibits at least TβRII. Therefore, insome embodiments, a TGFβ antagonist antibody, or combination ofantibodies, binds to at least TβRII. As used herein, a TβRII antibody(anti-TβRII antibody) generally refers to an antibody that binds toTβRII with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting TβRII. In certainembodiments, the extent of binding of an anti-TβRII antibody to anunrelated, non-TβRII protein is less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or less than about 1% of the binding of the antibody toTβRII as measured, for example, by a radioimmunoassay (RIA), Biacore, orother protein-protein interaction or binding affinity assay. In certainembodiments, an anti-TβRII antibody binds to an epitope of TβRII that isconserved among TβRII from different species. In certain preferredembodiments, an anti-TβRII antibody binds to human TβRII. In someembodiments, an anti-TβRII antibody may inhibit one or more ligands[e.g., TGFβ1; TGFβ2; TGFβ3; TGFβ1 and TGFβ3; TGFβ1 and TGFβ2; TGFβ2 andTGFβ3; or TGFβ1, TGFβ2, and TGFβ3] from binding to TβRII. In someembodiments, an anti-TβRII antibody is a multispecific antibody (e.g.,bi-specific antibody) that binds to TβRII and one or more ligands [e.g.,TGFβ1, TGFβ2, and TGFβ3], type I receptor (e.g., ALK5), and/orco-receptor (e.g., betaglycan). In some embodiments, the disclosurerelates to combinations of antibodies, and uses thereof, wherein thecombination of antibodies comprises an anti-TβRII antibody and one ormore additional antibodies that bind to, for example, one or moreligands [e.g., TGFβ1, TGFβ2, and TGFβ3], type I receptors (e.g., ALK5),and/or co-receptor (e.g., betaglycan).

In certain aspects, a TGFβ antagonist antibody, or combination ofantibodies, is an antibody that inhibits at least ALK5. Therefore, insome embodiments, a TGFβ antagonist antibody, or combination ofantibodies, binds to at least ALK5. As used herein, an ALK5 antibody(anti-ALK5 antibody) generally refers to an antibody that binds to ALK5with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting ALK5. In certainembodiments, the extent of binding of an anti-ALK5 antibody to anunrelated, non-ALK5 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or less than about 1% of the binding of the antibody to ALK5as measured, for example, by a radioimmunoassay (RIA), Biacore, or otherprotein-protein interaction or binding affinity assay. In certainembodiments, an anti-ALK5 antibody binds to an epitope of ALK5 that isconserved among ALK5 from different species. In certain preferredembodiments, an anti-ALK5 antibody binds to human ALK5. In someembodiments, an anti-ALK5 antibody may inhibit one or more ligands[e.g., TGFβ1; TGFβ2; TGFβ3; TGFβ1 and TGFβ3; TGFβ1 and TGFβ2; TGFβ2 andTGFβ3; or TGFβ1, TGFβ2, and TGFβ3] from binding to ALK5. In someembodiments, an anti-ALK5 antibody is a multispecific antibody (e.g.,bi-specific antibody) that binds to ALK5 and one or more ligands [e.g.,TGFβ1, TGFβ2, and TGFβ3], type II receptor (e.g., TβRII), and/orco-receptor (e.g., betaglycan). In some embodiments, the disclosurerelates to combinations of antibodies, and uses thereof, wherein thecombination of antibodies comprises an anti-ALK5 antibody and one ormore additional antibodies that bind to, for example, one or moreligands [e.g., TGFβ1, TGFβ2, and TGFβ3], type II receptors (e.g.,TβRII), and/or co-receptor (e.g., betaglycan).

In certain aspects, a TGFβ antagonist antibody, or combination ofantibodies, is an antibody that inhibits at least betaglycan. Therefore,in some embodiments, a TGFβ antagonist antibody, or combination ofantibodies, binds to at least betaglycan. As used herein, a betaglycanantibody (anti-betaglycan antibody) generally refers to an antibody thatbinds to betaglycan with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting betaglycan.In certain embodiments, the extent of binding of an anti-betaglycanantibody to an unrelated, non-betaglycan protein is less than about 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding ofthe antibody to betaglycan as measured, for example, by aradioimmunoassay (RIA), Biacore, or other protein-protein interaction orbinding affinity assay. In certain embodiments, an anti-betaglycanantibody binds to an epitope of betaglycan that is conserved amongbetaglycan from different species. In certain preferred embodiments, ananti-betaglycan antibody binds to human betaglycan. In some embodiments,an anti-betaglycan antibody may inhibit one or more ligands [e.g.,TGFβ1; TGFβ2; TGFβ3; TGFβ1 and TGFβ3; TGFβ1 and TGFβ2; TGFβ2 and TGFβ3;or TGFβ1, TGFβ2, and TGFβ3] from binding to betaglycan. In someembodiments, an anti-betaglycan antibody is a multispecific antibody(e.g., bi-specific antibody) that binds to betaglycan and one or moreligands [e.g., TGFβ1, TGFβ2, and TGFβ3], type I receptor (e.g., ALK5),and/or type II receptors (e.g., TβRII). In some embodiments, thedisclosure relates to combinations of antibodies, and uses thereof,wherein the combination of antibodies comprises an anti-betaglycanantibody and one or more additional antibodies that bind to, forexample, one or more ligands [e.g., TGFβ1, TGFβ2, and TGFβ3], type Ireceptors (e.g., ALK5), and/or type II receptors (e.g., TβRII).

The term antibody is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity. An antibody fragment refers to amolecule other than an intact antibody that comprises a portion of anintact antibody that binds the antigen to which the intact antibodybinds. Examples of antibody fragments include but are not limited to Fv,Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chainantibody molecules (e.g., scFv); and multispecific antibodies formedfrom antibody fragments. See, e.g., Hudson et al. (2003) Nat. Med.9:129-134; Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894, 5,587,458, and5,869,046. Antibodies disclosed herein may be polyclonal antibodies ormonoclonal antibodies. In certain embodiments, the antibodies of thepresent disclosure comprise a label attached thereto and able to bedetected (e.g., the label can be a radioisotope, fluorescent compound,enzyme, or enzyme co-factor). In preferred embodiments, the antibodiesof the present disclosure are isolated antibodies. Diabodies areantibody fragments with two antigen-binding sites that may be bivalentor bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudson et al.(2003) Nat. Med. 9:129-134 (2003); and Hollinger et al. (1993) Proc.Natl. Acad. Sci. USA 90: 6444-6448. Triabodies and tetrabodies are alsodescribed in Hudson et al. (2003) Nat. Med. 9:129-134. Single-domainantibodies are antibody fragments comprising all or a portion of theheavy-chain variable domain or all or a portion of the light-chainvariable domain of an antibody. In certain embodiments, a single-domainantibody is a human single-domain antibody. See, e.g., U.S. Pat. No.6,248,516. Antibody fragments can be made by various techniques,including but not limited to proteolytic digestion of an intact antibodyas well as production by recombinant host cells (e.g., E. coli orphage), as described herein.

The antibodies herein may be of any class. The class of an antibodyrefers to the type of constant domain or constant region possessed byits heavy chain. There are five major classes of antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), for example, IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, andIgA₂. The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are called alpha, delta, epsilon, gamma, andmu.

In general, an antibody for use in the methods disclosed hereinspecifically binds to its target antigen, preferably with high bindingaffinity. Affinity may be expressed as a K_(D) value and reflects theintrinsic binding affinity (e.g., with minimized avidity effects).Typically, binding affinity is measured in vitro, whether in a cell-freeor cell-associated setting. Any of a number of assays known in the art,including those disclosed herein, can be used to obtain binding affinitymeasurements including, for example, surface plasmon resonance (Biacore™assay), radiolabeled antigen binding assay (RIA), and ELISA. In someembodiments, antibodies of the present disclosure bind to their targetantigens (e.g. TGFβ1, TGFβ2, TGFβ2, ALK5, betaglycan, and TβRII.) withat least a K_(D) of 1×10⁻⁷ or stronger, 1×10⁻⁸ or stronger, 1×10⁻⁹ orstronger, 1×10⁻¹⁰ or stronger, 1×10⁻¹¹ or stronger, 1×10⁻¹² or stronger,1×10⁻¹³ or stronger, or 1×10⁻¹⁴ or stronger.

In certain embodiments, K_(D) is measured by RIA performed with the Fabversion of an antibody of interest and its target antigen as describedby the following assay. Solution binding affinity of Fabs for theantigen is measured by equilibrating Fab with a minimal concentration ofradiolabeled antigen (e.g., ¹²⁵I-labeled) in the presence of a titrationseries of unlabeled antigen, then capturing bound antigen with ananti-Fab antibody-coated plate [see, e.g., Chen et al. (1999) J. Mol.Biol. 293:865-881]. To establish conditions for the assay, multi-wellplates (e.g., MICROTITER® from Thermo Scientific) are coated (e.g.,overnight) with a capturing anti-Fab antibody (e.g., from Cappel Labs)and subsequently blocked with bovine serum albumin, preferably at roomtemperature (e.g., approximately 23° C.). In a non-adsorbent plate,radiolabeled antigen are mixed with serial dilutions of a Fab ofinterest [e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599]. The Fab ofinterest is then incubated, preferably overnight but the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation, preferably at room temperature for aboutone hour. The solution is then removed and the plate is washed timesseveral times, preferably with polysorbate 20 and PBS mixture. When theplates have dried, scintillant (e.g., MICROSCINT® from Packard) isadded, and the plates are counted on a gamma counter (e.g., TOPCOUNT®from Packard).

According to another embodiment, K_(D) is measured using surface plasmonresonance assays using, for example a BIACORE® 2000 or a BIACORE® 3000(Biacore, Inc., Piscataway, N.J.) with immobilized antigen CM5 chips atabout 10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, Biacore, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions. Forexample, an antigen can be diluted with 10 mM sodium acetate, pH 4.8, to5 pg/ml (about 0.2 pM) before injection at a flow rate of 5 μl/minute toachieve approximately 10 response units (RU) of coupled protein.Following the injection of antigen, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%polysorbate 20 (TWEEN-20®) surfactant (PBST) at at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using, for example, a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on) [see, e.g., Chen et al., (1999) J. Mol. Biol.293:865-881]. If the on-rate exceeds, for example, 10⁶ M⁻¹ s⁻¹ by thesurface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (e.g.,excitation=295 nm; emission=340 nm, 16 nm band-pass) of a 20 nManti-antigen antibody (Fab form) in PBS in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO® spectrophotometer (ThermoSpectronic) with a stirred cuvette.

The nucleic acid and amino acid sequences of TβRII, ALK5, betaglycan,TGFβ1, TGFβ2, and TGFβ3, particularly human sequences, are well known inthe art and thus antibody antagonists for use in accordance with thisdisclosure may be routinely made by the skilled artisan based on theknowledge in the art and teachings provided herein.

In certain embodiments, an antibody provided herein is a chimericantibody. A chimeric antibody refers to an antibody in which a portionof the heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species. Certain chimeric antibodies aredescribed, for example, in U.S. Pat. No. 4,816,567; and Morrison et al.,(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855. In some embodiments, achimeric antibody comprises a non-human variable region (e.g., avariable region derived from a mouse, rat, hamster, rabbit, or non-humanprimate, such as a monkey) and a human constant region. In someembodiments, a chimeric antibody is a “class switched” antibody in whichthe class or subclass has been changed from that of the parent antibody.In general, chimeric antibodies include antigen-binding fragmentsthereof.

In certain embodiments, a chimeric antibody provided herein is ahumanized antibody. A humanized antibody refers to a chimeric antibodycomprising amino acid residues from non-human hypervariable regions(HVRs) and amino acid residues from human framework regions (FRs). Incertain embodiments, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the HVRs (e.g., CDRs) correspond to those of anon-human antibody, and all or substantially all of the FRs correspondto those of a human antibody. A humanized antibody optionally maycomprise at least a portion of an antibody constant region derived froma human antibody. A “humanized form” of an antibody, e.g., a non-humanantibody, refers to an antibody that has undergone humanization.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633 andare further described, for example, in Riechmann et al., (1988) Nature332:323-329; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri et al., (2005) Methods 36:25-34 [describing SDR(a-CDR) grafting]; Padlan, Mol. Immunol. (1991) 28:489-498 (describing“resurfacing”); Dall'Acqua et al. (2005) Methods 36:43-60 (describing“FR shuffling”); Osbourn et al. (2005) Methods 36:61-68; and Klimka etal. Br. J. Cancer (2000) 83:252-260 (describing the “guided selection”approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method [see, e.g., Sims et al. (1993) J. Immunol. 151:2296]; frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light-chain or heavy-chain variable regions [see,e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; andPresta et al. (1993) J. Immunol., 151:2623]; human mature (somaticallymutated) framework regions or human germline framework regions [see,e.g., Almagro and Fransson (2008) Front. Biosci. 13:1619-1633]; andframework regions derived from screening FR libraries [see, e.g., Bacaet cd., (1997) J. Biol. Chem. 272:10678-10684; and Rosok et cd., (1996)J. Biol. Chem. 271:22611-22618].

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel (2001) Curr. Opin. Pharmacol. 5: 368-74 and Lonberg (2008) Curr.Opin. Immunol. 20:450-459.

Human antibodies may be prepared by administering an immunogen (e.g aTβRII, ALK5, betaglycan, TGFβ1, TGFβ2, or TGFβ3 polypeptide) to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicanimals, the endogenous immunoglobulin loci have generally beeninactivated. For a review of methods for obtaining human antibodies fromtransgenic animals, see, for example, Lonberg (2005) Nat. Biotechnol.23:1117-1125; U.S. Pat. Nos. 6,075,181 and 6,150,584 (describingXENOMOUSE™ technology); U.S. Pat. No. 5,770,429 (describing HuMab®technology); U.S. Pat. No. 7,041,870 (describing K-M MOUSE® technology);and U.S. Patent Application Publication No. 2007/0061900 (describingVelociMouse® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, for example, bycombining with a different human constant region.

Human antibodies provided herein can also be made by hybridoma-basedmethods. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described [see,e.g., Kozbor J. Immunol., (1984) 133: 3001; Brodeur et al. (1987)Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York; and Boerner et al. (1991) J. Immunol.,147: 86]. Human antibodies generated via human B-cell hybridomatechnology are also described in Li et al., (2006) Proc. Natl. Acad.Sci. USA, 103:3557-3562. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 (describing production of monoclonalhuman IgM antibodies from hybridoma cell lines) and Ni, XiandaiMianyixue (2006) 26(4):265-268 (2006) (describing human-humanhybridomas). Human hybridoma technology (Trioma technology) is alsodescribed in Vollmers and Brandlein (2005) Histol. Histopathol.,20(3):927-937 (2005) and Vollmers and Brandlein (2005) Methods Find Exp.Clin. Pharmacol., 27(3):185-91.

Human antibodies provided herein may also be generated by isolating Fvclone variable-domain sequences selected from human-derived phagedisplay libraries. Such variable-domain sequences may then be combinedwith a desired human constant domain. Techniques for selecting humanantibodies from antibody libraries are described herein.

For example, antibodies of the present disclosure may be isolated byscreening combinatorial libraries for antibodies with the desiredactivity or activities. A variety of methods are known in the art forgenerating phage-display libraries and screening such libraries forantibodies possessing the desired binding characteristics. Such methodsare reviewed, for example, in Hoogenboom et al. (2001) in Methods inMolecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa,N.J. and further described, for example, in the McCafferty et al. (1991)Nature 348:552-554; Clackson et al., (1991) Nature 352: 624-628; Markset al. (1992) J. Mol. Biol. 222:581-597; Marks and Bradbury (2003) inMethods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa,N.J.; Sidhu et al. (2004) J. Mol. Biol. 338(2):299-310; Lee et al.(2004) J. Mol. Biol. 340(5):1073-1093; Fellouse (2004) Proc. Natl. Acad.Sci. USA 101(34):12467-12472; and Lee et al. (2004) J. Immunol. Methods284(1-2): 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. (1994) Ann. Rev.Immunol., 12: 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen (e.g., a TβRII, TGFβ1, TGFβ2, or TGFβ3 polypeptide) withoutthe requirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned (e.g., from human) to provide a single sourceof antibodies directed against a wide range of non-self and alsoself-antigens without any immunization as described by Griffiths et al.(1993) EMBO J, 12: 725-734. Finally, naive libraries can also be madesynthetically by cloning un-rearranged V-gene segments from stem cellsand using PCR primers containing random sequence to encode the highlyvariable CDR3 regions and to accomplish rearrangement in vitro, asdescribed by Hoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388.Patent publications describing human antibody phage libraries include,for example: U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

In certain embodiments, an antibody provided herein is a multispecificantibody, for example, a bispecific antibody. Multispecific antibodies(typically monoclonal antibodies) have binding specificities for atleast two different epitopes (e.g., two, three, four, five, or six ormore) on one or more (e.g., two, three, four, five, six or more)antigens.

Engineered antibodies with three or more functional antigen bindingsites, including “octopus antibodies,” are also included herein (see,e.g., US 2006/0025576A1).

In certain embodiments, the antibodies disclosed herein are monoclonalantibodies. Monoclonal antibody refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical and/or bind the sameepitope, except for possible variant antibodies, e.g., containingnaturally occurring mutations or arising during production of amonoclonal antibody preparation, such variants generally being presentin minor amounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentepitopes, each monoclonal antibody of a monoclonal antibody preparationis directed against a single epitope on an antigen. Thus, the modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present methods may be made by a variety of techniques,including but not limited to the hybridoma method, recombinant DNAmethods, phage-display methods, and methods utilizing transgenic animalscontaining all or part of the human immunoglobulin loci, such methodsand other exemplary methods for making monoclonal antibodies beingdescribed herein.

For example, by using immunogens derived from TβRII,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols [see, e.g., Antibodies: A Laboratory Manual (1988)ed. by Harlow and Lane, Cold Spring Harbor Press]. A mammal, such as amouse, hamster, or rabbit can be immunized with an immunogenic form ofthe TβRII polypeptide, an antigenic fragment which is capable ofeliciting an antibody response, or a fusion protein. Techniques forconferring immunogenicity on a protein or peptide include conjugation tocarriers or other techniques well known in the art. An immunogenicportion of a TβRII polypeptide can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassayscan be used with the immunogen as antigen to assess the levels ofantibody production and/or level of binding affinity.

Following immunization of an animal with an antigenic preparation ofTβRII, antisera can be obtained and, if desired, polyclonal antibodiescan be isolated from the serum. To produce monoclonal antibodies,antibody-producing cells (lymphocytes) can be harvested from animmunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, and include, for example, thehybridoma technique [see, e.g., Kohler and Milstein (1975) Nature, 256:495-497], the human B cell hybridoma technique [see, e.g., Kozbar et al.(1983) Immunology Today, 4:72], and the EBV-hybridoma technique toproduce human monoclonal antibodies [Cole et al. (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96]. Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with a TβRII polypeptide, and monoclonalantibodies isolated from a culture comprising such hybridoma cells.

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein therebygenerating an Fc-region variant. The Fc-region variant may comprise ahuman Fc-region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g., a substitution,deletion, and/or addition) at one or more amino acid positions.

For example, the present disclosure contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet for which certain effector functions[e.g., complement-dependent cytotoxicity (CDC) and antibody-dependentcellular cytotoxicity (ADCC)] are unnecessary or deleterious. In vitroand/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in, forexample, Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362;Hellstrom, I. et al. (1986) Proc. Nat'l Acad. Sci. USA 83:7059-7063;Hellstrom, I et al. (1985) Proc. Nat'l Acad. Sci. USA 82:1499-1502; U.S.Pat. No. 5,821,337; and Bruggemann, M. et al. (1987) J. Exp. Med.166:1351-1361. Alternatively, non-radioactive assay methods may beemployed (e.g., ACTI™, non-radioactive cytotoxicity assay for flowcytometry; CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay, Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, for example, in an animal model such as that disclosed inClynes et al. (1998) Proc. Nat'l Acad. Sci. USA 95:652-656. C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity [see, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402]. To assess complementactivation, a CDC assay may be performed [see, e.g., Gazzano-Santoro etal. (1996) J. Immunol. Methods 202:163; Cragg, M. S. et al. (2003) Blood101:1045-1052; and Cragg, M. S, and M. J. Glennie (2004) Blood103:2738-2743]. FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art[see, e.g., Petkova, S. B. et al. (2006) Int. Immunol.18(12):1759-1769].

Antibodies of the present disclosure with reduced effector functioninclude those with substitution of one or more of Fc region residues238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

In certain embodiments, it may be desirable to createcysteine-engineered antibodies, e.g., “thioMAbs,” in which one or moreresidues of an antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to other moieties,such as drug moieties or linker-drug moieties, to create animmunoconjugate, as described further herein. In certain embodiments,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering)of the heavy chain; and S400 (EU numbering) of the heavy-chain Fcregion. Cysteine engineered antibodies may be generated as described,for example, in U.S. Pat. No. 7,521,541.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore™ binding assay, Biacore AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain embodiments, amino acid sequence variants of the antibodiesand/or the binding polypeptides provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody and/or binding polypeptide.Amino acid sequence variants of an antibody and/or binding polypeptidesmay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody and/or binding polypeptide, orby peptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into, and/or substitutions of residues within,the amino acid sequences of the antibody and/or binding polypeptide. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., target-binding (TβRII,ALK5, betaglycan, TGFβ1, TGFβ2, and/or TGFβ3).

Alterations (e.g., substitutions) may be made in HVRs, for example, toimprove antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process (see, e.g.,Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs(a-CDRs), with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described in the art [see, e.g., Hoogenboomet al., in Methods in Molecular Biology 178:1-37, O'Brien et al., ed.,Human Press, Totowa, N.J., (2001)]. In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized. HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind to the antigen.For example, conservative alterations (e.g., conservative substitutionsas provided herein) that do not substantially reduce binding affinitymay be made in HVRs. Such alterations may be outside of HVR “hotspots”or SDRs. In certain embodiments of the variant VH and VL sequencesprovided above, each HVR either is unaltered, or contains no more thanone, two, or three amino acid substitutions.

A useful method for identification of residues or regions of theantibody and/or the binding polypeptide that may be targeted formutagenesis is called “alanine scanning mutagenesis”, as described byCunningham and Wells (1989) Science, 244:1081-1085. In this method, aresidue or group of target residues (e.g., charged residues such as arg,asp, his, lys, and glu) are identified and replaced by a neutral ornegatively charged amino acid (e.g., alanine or polyalanine) todetermine whether the interaction of the antibody or binding polypeptidewith antigen is affected. Further substitutions may be introduced at theamino acid locations demonstrating functional sensitivity to the initialsubstitutions. Alternatively, or additionally, a crystal structure of anantigen-antibody complex can be used to identify contact points betweenthe antibody and antigen. Such contact residues and neighboring residuesmay be targeted or eliminated as candidates for substitution. Variantsmay be screened to determine whether they contain the desiredproperties.

Amino-acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include fusion of the N- or C-terminusof the antibody to an enzyme (e.g., for ADEPT) or a polypeptide whichincreases the serum half-life of the antibody.

In certain embodiments, an antibody and/or binding polypeptide providedherein may be further modified to contain additional non-proteinaceousmoieties that are known in the art and readily available. The moietiessuitable for derivatization of the antibody and/or binding polypeptideinclude but are not limited to water-soluble polymers. Non-limitingexamples of water-soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody and/orbinding polypeptide may vary, and if more than one polymer are attached,they can be the same or different molecules. In general, the numberand/or type of polymers used for derivatization can be determined basedon considerations including, but not limited to, the particularproperties or functions of the antibody and/or binding polypeptide to beimproved, whether the antibody derivative and/or binding polypeptidederivative will be used in a therapy under defined conditions.

D. Small Molecules

In certain aspects, a TGFβ antagonist to be used in accordance with themethods and uses disclosed herein is a small molecule (TGFβ antagonistsmall molecule) or combination of small molecules. A TGFβ antagonistsmall molecule, or combination of small molecules, may inhibit, forexample, one or more ligands (e.g., TGFβ1, TGFβ2, and TGFβ3), TβRIIreceptor, TβRII-associated type I receptor (e.g., ALK5),TβRII-associated co-receptor (e.g., betaglycan), and/or downstreamsignaling component (e.g., Smads). In some embodiments, the ability fora TGFβ antagonist small molecule, or combination of small molecules, toinhibit signaling (e.g., Smad signaling) is determined in a cell-basedassay including, for example, those described herein. A TGFβ antagonistsmall molecule, or combination of small molecules, may be used alone orin combination with one or more additional supportive therapies oractive agents to treat heterotopic ossification, preferably preventingor reducing the severity or duration of one or more complications ofheterotopic ossification.

In certain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits at least TGFβ1 (e.g., inhibition of Smadsignaling). Therefore, in some embodiments, a small molecule inhibitorof TGFβ1 binds to TGFβ1. In some embodiments, a small molecule inhibitorof TGFβ1 inhibits expression (e.g., transcription, translation,secretion, or combinations thereof) of TGFβ1. In some embodiments, asmall molecule inhibitor of TGFβ1 further inhibits one or more of TGFβ2,TGFβ3, TβRII, ALK5, and betaglycan. In some embodiments, a smallmolecule inhibitor of TGFβ1 does not inhibit or does not substantiallyinhibit TGFβ2. In some embodiments, a small molecule inhibitor of TGFβ1further inhibits TGFβ3 but does not inhibit or does not substantiallyinhibit TGFβ2. In certain aspects, a TGFβ antagonist small molecule, orcombination of small molecules, inhibits at least TGFβ2 (e.g.,inhibition of Smad signaling). Therefore, in some embodiments, a smallmolecule inhibitor of TGFβ2 binds to TGFβ2. In some embodiments, a smallmolecule inhibitor of TGFβ2 inhibits expression (e.g., transcription,translation, secretion, or combinations thereof) of TGFβ2. In someembodiments, a small molecule inhibitor of TGFβ2 further inhibits one ormore of TGFβ3, TGFβ1, TβRII, ALK5, and betaglycan. In certain aspects, aTGFβ antagonist small molecule, or combination of small molecules,inhibits at least TGFβ3 (e.g., inhibition of Smad signaling). Therefore,in some embodiments, a small molecule inhibitor of TGFβ3 binds to TGFβ3.In some embodiments, a small molecule inhibitor of TGFβ3 inhibitsexpression (e.g., transcription, translation, secretion, or combinationsthereof) of TGFβ3. In some embodiments, a small molecule inhibitor ofTGFβ3 further inhibits one or more of TGFβ2, TGFβ1, TβRII, ALK5, andbetaglycan. In some embodiments, a small molecule inhibitor of TGFβ3does not inhibit or does not substantially inhibit TGFβ2. In someembodiments, a small molecule inhibitor of TGFβ3 further inhibits TGFβ1but does not inhibit or does not substantially inhibit TGFβ2. In certainaspects, a TGFβ antagonist small molecule, or combination of smallmolecules, inhibits at least TβRII (e.g., inhibition of Smad signaling).Therefore, in some embodiments, a small molecule inhibitor of TβRIIbinds to TβRII. In some embodiments, a small molecule inhibitor of TβRIIinhibits expression (e.g., transcription, translation, secretion, orcombinations thereof) of TβRII. In some embodiments, a small moleculeinhibitor of TβRII further inhibits one or more of TGFβ1, TGFβ2, TGFβ3,ALK5, and betaglycan. In certain aspects, a TGFβ antagonist smallmolecule, or combination of small molecules, inhibits TGFβ1 from bindingto TβRII. In certain aspects, a TGFβ antagonist small molecule, orcombination of small molecules, inhibits TGFβ2 from binding to TβRII. Incertain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ3 from binding to TβRII. In certainaspects, a TGFβ antagonist small molecule, or combination of smallmolecules, inhibits TGFβ1 and TGFβ3 from binding to TβRII. In certainaspects, a TGFβ antagonist small molecule, or combination of smallmolecules, inhibits TGFβ1, TGFβ2, and TGFβ3 from binding to TβRII. Incertain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ1 from binding to TβRII but does notinhibit or does not substantially inhibit TGFβ2 from binding to TβRII.In certain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ3 from binding to TβRII but does notinhibit or does not substantially inhibit TGFβ2 from binding to TβRII.In certain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ1 and TGFβ3 from binding to TβRII but doesnot inhibit or does not substantially inhibit TGFβ2 from binding toTβRII. In certain aspects, a Tβ TGFβ RII antagonist small molecule, orcombination of small molecules, inhibits at least ALK5 (e.g., inhibitionof Smad signaling). Therefore, in some embodiments, a small moleculeinhibitor of ALK5 binds to ALK5. In some embodiments, a small moleculeinhibitor of ALK5 inhibits expression (e.g., transcription, translation,secretion, or combinations thereof) of ALK5. In some embodiments, asmall molecule inhibitor of ALK5 further inhibits one or more of TGFβ1,TGFβ2, TGFβ3, TβRII, and betaglycan. In certain aspects, a TGFβantagonist small molecule, or combination of small molecules, inhibitsTGFβ1 from binding to ALK5. In certain aspects, a TGFβ antagonist smallmolecule, or combination of small molecules, inhibits TGFβ2 from bindingto ALK5. In certain aspects, a TGFβ antagonist small molecule, orcombination of small molecules, inhibits TGFβ3 from binding to ALK5. Incertain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ1 and TGFβ3 from binding to ALK5. Incertain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ1, TGFβ2, and TGFβ3 from binding to ALK5.In certain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ1 from binding to ALK5 but does notinhibit or does not substantially inhibit TGFβ2 from binding to ALK5. Incertain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ3 from binding to ALK5 but does notinhibit or does not substantially inhibit TGFβ2 from binding to ALK5. Incertain aspects, a TGFβ antagonist small molecule, or combination ofsmall molecules, inhibits TGFβ1 and TGFβ3 from binding to ALK5 but doesnot inhibit or does not substantially inhibit TGFβ2 from binding toALK5. In certain aspects, a TGFβ antagonist small molecule, orcombination of small molecules, inhibits at least betaglycan (e.g.,inhibition of Smad signaling). Therefore, in some embodiments, a smallmolecule inhibitor of betaglycan binds to betaglycan. In someembodiments, a small molecule inhibitor of betaglycan inhibitsexpression (e.g., transcription, translation, secretion, or combinationsthereof) of betaglycan. In some embodiments, a small molecule inhibitorof betaglycan further inhibits one or more of TGFβ1, TGFβ2, TGFβ3,TβRII, and ALK5. In certain aspects, a TGFβ antagonist small molecule,or combination of small molecules, inhibits TGFβ1 from binding tobetaglycan. In certain aspects, a TGFβ antagonist small molecule, orcombination of small molecules, inhibits TGFβ2 from binding tobetaglycan. In certain aspects, a TGFβ antagonist small molecule, orcombination of small molecules, inhibits TGFβ3 from binding tobetaglycan. In certain aspects, a TGFβ antagonist small molecule, orcombination of small molecules, inhibits TGFβ1 and TGFβ3 from binding tobetaglycan. In certain aspects, a TGFβ antagonist small molecule, orcombination of small molecules, inhibits TGFβ1, TGFβ2, and TGFβ3 frombinding to betaglycan. In certain aspects, a TGFβ antagonist smallmolecule, or combination of small molecules, inhibits TGFβ1 from bindingto betaglycan but does not inhibit or does not substantially inhibitTGFβ2 from binding to betaglycan. In certain aspects, a TGFβ antagonistsmall molecule, or combination of small molecules, inhibits TGFβ3 frombinding to betaglycan but does not inhibit or does not substantiallyinhibit TGFβ2 from binding to betaglycan. In certain aspects, a TGFβantagonist small molecule, or combination of small molecules, inhibitsTGFβ1 and TGFβ3 from binding to betaglycan but does not inhibit or doesnot substantially inhibit TGFβ2 from binding to betaglycan.

TGFβ antagonist small molecules can be direct or indirect inhibitors.For example, a TGFβ antagonist small molecule, or combination of smallmolecules, may inhibit the expression (e.g., transcription, translation,cellular secretion, or combinations thereof) of at least one or more ofTβRII, ALK5, betaglycan, TGFβ1, TGFβ2, TGFβ3, and/or one or moredownstream signaling factors (Smads). Alternatively, a direct TGFβantagonist small molecule, or combination of small molecules, maydirectly bind to, for example, one or more of TβRII, ALK5, betaglycan,TGFβ1, TGFβ2, and TGFβ3 or one or more downstream signaling factors.Combinations of one or more indirect and one or more direct TGFβantagonist small molecule may be used in accordance with the methodsdisclosed herein.

Binding organic small molecule antagonists of the present disclosure maybe identified and chemically synthesized using known methodology (see,e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). In general,small molecule antagonists of the disclosure are usually less than about2000 daltons in size, alternatively less than about 1500, 750, 500, 250or 200 daltons in size, wherein such organic small molecules that arecapable of binding, preferably specifically, to a polypeptide asdescribed herein (e.g., TβRII, ALK5, betaglycan, TGFβ1, TGFβ2, andTGFβ3). Such small molecule antagonists may be identified without undueexperimentation using well-known techniques. In this regard, it is notedthat techniques for screening organic small molecule libraries formolecules that are capable of binding to a polypeptide target arewell-known in the art (see, e.g., international patent publication Nos.WO00/00823 and WO00/39585).

Binding organic small molecules of the present disclosure may be, forexample, aldehydes, ketones, oximes, hydrazones, semicarbazones,carbazides, primary amines, secondary amines, tertiary amines,N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,thioethers, disulfides, carboxylic acids, esters, amides, ureas,carbamates, carbonates, ketals, thioketals, acetals, thioacetals, arylhalides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds, and acid chlorides.

E. Nucleic Acids

In certain aspects, a TGFβ antagonist to be used in accordance with themethods and uses disclosed herein is a polynucleotide (TGFβ antagonistpolynucleotide) or combination of polynucleotides. A TGFβ antagonistpolynucleotide, or combination of polynucleotides, may inhibit, forexample, one or more ligands (e.g., TGFβ1, TGFβ2, and TGFβ3), TβRIIreceptor, TβRII-associated type I receptor (e.g., ALK5),TβRII-associated co-receptor (e.g., betaglycan), and/or downstreamsignaling component (e.g., Smads). In some embodiments, the ability fora TGFβ antagonist polynucleotide, or combination of polynucleotides, toinhibit signaling (e.g., Smad signaling) is determined in a cell-basedassay including, for example, those described herein. A TGFβ antagonistpolynucleotide, or combination of polynucleotide, may be used, alone orin combination with one or more additional supportive therapies oractive agents, to treat heterotopic ossification, preferably preventingor reducing the severity or duration of one or more complications ofheterotopic ossification.

In certain aspects, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits at least TGFβ1 (e.g., inhibition of Smadsignaling). Therefore, in some embodiments, a polynucleotide inhibitorof TGFβ1 binds to TGFβ1. In some embodiments, a polynucleotide inhibitorof TGFβ1 inhibits expression (e.g., transcription, translation,secretion, or combinations thereof) of TGFβ1. In some embodiments, apolynucleotide inhibitor of TGFβ1 further inhibits one or more of TGFβ2,TGFβ3, TβRII, ALK5, and betaglycan. In some embodiments, apolynucleotide inhibitor of TGFβ1 does not inhibit or does notsubstantially inhibit TGFβ2. In some embodiments, a polynucleotideinhibitor of TGFβ1 further inhibits TGFβ3 but does not inhibit or doesnot substantially inhibit TGFβ2. In certain aspects, a TGFβ antagonistpolynucleotide, or combination of polynucleotides, inhibits at leastTGFβ2 (e.g., inhibition of Smad signaling). Therefore, in someembodiments, a polynucleotide inhibitor of TGFβ2 binds to TGFβ2. In someembodiments, a polynucleotide inhibitor of TGFβ2 inhibits expression(e.g., transcription, translation, secretion, or combinations thereof)of TGFβ2. In some embodiments, a polynucleotide inhibitor of TGFβ2further inhibits one or more of TGFβ3, TGFβ1, TβRII, ALK5, andbetaglycan. In certain aspects, a TGFβ antagonist polynucleotide, orcombination of polynucleotides, inhibits at least TGFβ3 (e.g.,inhibition of Smad signaling). Therefore, in some embodiments, apolynucleotide inhibitor of TGFβ3 binds to TGFβ3. In some embodiments, apolynucleotide inhibitor of TGFβ3 inhibits expression (e.g.,transcription, translation, secretion, or combinations thereof) ofTGFβ3. In some embodiments, a polynucleotide inhibitor of TGFβ3 furtherinhibits one or more of TGFβ2, TGFβ1, TβRII, ALK5, and betaglycan. Insome embodiments, a polynucleotide inhibitor of TGFβ3 does not inhibitor does not substantially inhibit TGFβ2. In some embodiments, apolynucleotide inhibitor of TGFβ3 further inhibits TGFβ1 but does notinhibit or does not substantially inhibit TGFβ2. In certain aspects, aTGFβ antagonist polynucleotide, or combination of polynucleotides,inhibits at least TβRII (e.g., inhibition of Smad signaling). Therefore,in some embodiments, a polynucleotide inhibitor of TβRII binds to TβRII.In some embodiments, a polynucleotide inhibitor of TβRII inhibitsexpression (e.g., transcription, translation, secretion, or combinationsthereof) of TβRII. In some embodiments, a polynucleotide inhibitor ofTβRII further inhibits one or more of TGFβ1, TGFβ2, TGFβ3, ALK5, andbetaglycan. In some embodiments, a TGFβ antagonist polynucleotide, orcombination of polynucleotides, inhibits TGFβ1 from binding to TβRII. Insome embodiments a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ2 from binding to TβRII. In someembodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ3 from binding to TβRII. In someembodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1 and TGFβ3 from binding to TβRII. In someembodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1, TGFβ2, and TGFβ3 from binding to TβRII.In some embodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1 from binding to TβRII but does notinhibit or does not substantially inhibit TGFβ2 from binding to TβRII.In some embodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ3 from binding to TβRII but does notinhibit or does not substantially inhibit TGFβ2 from binding to TβRII.In some embodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1 and TGFβ3 from binding to TβRII but doesnot inhibit or does not substantially inhibit TGFβ2 from binding toTβRII. In certain aspects, a TGFβ antagonist polynucleotide, orcombination of polynucleotides, inhibits at least ALK5 (e.g., inhibitionof Smad signaling). Therefore, in some embodiments, a polynucleotideinhibitor of ALK5 binds to ALK5. In some embodiments, a polynucleotideinhibitor of ALK5 inhibits expression (e.g., transcription, translation,secretion, or combinations thereof) of ALK5. In some embodiments, apolynucleotide inhibitor of ALK5 further inhibits one or more of TGFβ1,TGFβ2, TGFβ3, TβRII, and betaglycan. In some embodiments, a TGFβantagonist polynucleotide, or combination of polynucleotides, inhibitsTGFβ1 from binding to ALK5. In some embodiments, a TGFβ antagonistpolynucleotide, or combination of polynucleotides, inhibits TGFβ2 frombinding to ALK5. In some embodiments, a TGFβ antagonist polynucleotide,or combination of polynucleotides, inhibits TGFβ3 from binding to ALK5.In some embodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1 and TGFβ3 from binding to ALK5. In someembodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1, TGFβ2, and TGFβ3 from binding to ALK5.In some embodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1 from binding to ALK5 but does notinhibit or does not substantially inhibit TGFβ2 from binding to ALK5. Insome embodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ3 from binding to ALK5 but does notinhibit or does not substantially inhibit TGFβ2 from binding to ALK5. Insome embodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1 and TGFβ3 from binding to ALK5 but doesnot inhibit or does not substantially inhibit TGFβ2 from binding toALK5. In certain aspects, a TGFβ antagonist polynucleotide, orcombination of polynucleotides, inhibits at least betaglycan (e.g.,inhibition of Smad signaling). Therefore, in some embodiments, apolynucleotide inhibitor of betaglycan binds to betaglycan. In someembodiments, a polynucleotide inhibitor of betaglycan inhibitsexpression (e.g., transcription, translation, secretion, or combinationsthereof) of betaglycan. In some embodiments, a polynucleotide inhibitorof betaglycan further inhibits one or more of TGFβ1, TGFβ2, TGFβ3,TβRII, and ALK5. In some embodiments, a TGFβ antagonist polynucleotide,or combination of polynucleotides, inhibits TGFβ1 from binding tobetaglycan. In some embodiments, a TGFβ antagonist polynucleotide, orcombination of polynucleotides, inhibits TGFβ2 from binding tobetaglycan. In some embodiments, a TGFβ antagonist polynucleotide, orcombination of polynucleotides, inhibits TGFβ3 from binding tobetaglycan. In some embodiments, a TGFβ antagonist polynucleotide, orcombination of polynucleotides, inhibits TGFβ1 and TGFβ3 from binding tobetaglycan. In some embodiments, a TGFβ antagonist polynucleotide, orcombination of polynucleotides, inhibits TGFβ1, TGFβ2, and TGFβ3 frombinding to betaglycan. In some embodiments, a TGFβ antagonistpolynucleotide, or combination of polynucleotides, inhibits TGFβ1 frombinding to betaglycan but does not inhibit or does not substantiallyinhibit TGFβ2 from binding to betaglycan. In some embodiments, a TGFβantagonist polynucleotide, or combination of polynucleotides, inhibitsTGFβ3 from binding to betaglycan but does not inhibit or does notsubstantially inhibit TGFβ2 from binding to betaglycan. In someembodiments, a TGFβ antagonist polynucleotide, or combination ofpolynucleotides, inhibits TGFβ1 and TGFβ3 from binding to betaglycan butdoes not inhibit or does not substantially inhibit TGFβ2 from binding tobetaglycan.

The polynucleotide antagonists of the present disclosure may be anantisense nucleic acid, an RNAi molecule [e.g., small interfering RNA(siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)], an aptamer and/ora ribozyme. The nucleic acid and amino acid sequences of human TβRII,ALK5, betaglycan, TGFβ1, TGFβ2, and TGFβ3 are known in the art and thuspolynucleotide antagonists for use in accordance with methods of thepresent disclosure may be routinely made by the skilled artisan based onthe knowledge in the art and teachings provided herein.

For example, antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed, for example, in Okano (1991) J.Neurochem. 56:560; Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Cooney et al. (1988) Science 241:456; andDervan et al., (1991)Science 251:1300. The methods are based on bindingof a polynucleotide to a complementary DNA or RNA. In some embodiments,the antisense nucleic acids comprise a single-stranded RNA or DNAsequence that is complementary to at least a portion of an RNAtranscript of a desired gene. However, absolute complementarity,although preferred, is not required.

A sequence “complementary to at least a portion of an RNA,” referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids of a gene disclosed herein, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the larger the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Polynucleotides that are complementary to the 5′ end of the message, forexample, the 5′-untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′-untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well [see, e.g., Wagner, R., (1994) Nature372:333-335]. Thus, oligonucleotides complementary to either the 5′- or3′-untranslated, noncoding regions of a gene of the disclosure, could beused in an antisense approach to inhibit translation of an endogenousmRNA. Polynucleotides complementary to the 5′-untranslated region of themRNA should include the complement of the AUG start codon. Antisensepolynucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could be used in accordance with themethods of the present disclosure. Whether designed to hybridize to the5′-untranslated, 3′-untranslated, or coding regions of an mRNA of thedisclosure, antisense nucleic acids should be at least six nucleotidesin length, and are preferably oligonucleotides ranging from 6 to about50 nucleotides in length. In specific aspects, the oligonucleotide is atleast 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides,or at least 50 nucleotides.

In one embodiment, the antisense nucleic acid of the present disclosureis produced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of a gene of the disclosure. Such a vectorwould contain a sequence encoding the desired antisense nucleic acid.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding desired genes of the instantdisclosure, or fragments thereof, can be by any promoter known in theart to act in vertebrate, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include, but are not limitedto, the SV40 early promoter region [see, e.g., Benoist and Chambon(1981) Nature 29:304-310], the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus [see, e.g., Yamamoto et al. (1980)Cell 22:787-797], the herpes thymidine promoter [see, e.g., Wagner etal. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445], and theregulatory sequences of the metallothionein gene [see, e.g., Brinster,et al. (1982) Nature 296:39-42].

In some embodiments, the polynucleotide antagonists are interfering RNAor RNAi molecules that target the expression of one or more genes. RNAirefers to the expression of an RNA which interferes with the expressionof the targeted mRNA. Specifically, RNAi silences a targeted gene viainteracting with the specific mRNA through a siRNA (small interferingRNA). The ds RNA complex is then targeted for degradation by the cell.An siRNA molecule is a double-stranded RNA duplex of 10 to 50nucleotides in length, which interferes with the expression of a targetgene which is sufficiently complementary (e.g. at least 80% identity tothe gene). In some embodiments, the siRNA molecule comprises anucleotide sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100%identical to the nucleotide sequence of the target gene.

Additional RNAi molecules include short-hairpin RNA (shRNA); alsoshort-interfering hairpin and microRNA (miRNA). The shRNA moleculecontains sense and antisense sequences from a target gene connected by aloop. The shRNA is transported from the nucleus into the cytoplasm, andit is degraded along with the mRNA. Pol III or U6 promoters can be usedto express RNAs for RNAi. Paddison et al. [Genes & Dev. (2002)16:948-958, 2002] have used small RNA molecules folded into hairpins asa means to effect RNAi. Accordingly, such short hairpin RNA (shRNA)molecules are also advantageously used in the methods described herein.The length of the stem and loop of functional shRNAs varies; stemlengths can range anywhere from about 25 to about 30 nt, and loop sizecan range between 4 to about 25 nt without affecting silencing activity.While not wishing to be bound by any particular theory, it is believedthat these shRNAs resemble the double-stranded RNA (dsRNA) products ofthe DICER RNase and, in any event, have the same capacity for inhibitingexpression of a specific gene. The shRNA can be expressed from alentiviral vector. An miRNA is a single-stranded RNA of about 10 to 70nucleotides in length that are initially transcribed as pre-miRNAcharacterized by a “stem-loop” structure and which are subsequentlyprocessed into mature miRNA after further processing through the RISC.

Molecules that mediate RNAi, including without limitation siRNA, can beproduced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199,2002), hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase(Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc NatlAcad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-strandedRNA using a nuclease such as E. coli RNase III (Yang et al., Proc NatlAcad Sci USA 99:9942-9947, 2002).

According to another aspect, the disclosure provides polynucleotideantagonists including but not limited to, a decoy DNA, a double-strandedDNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, aviral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA,a double-stranded RNA, a molecule capable of generating RNAinterference, or combinations thereof.

In some embodiments, the polynucleotide antagonists of the disclosureare aptamers. Aptamers are nucleic acid molecules, includingdouble-stranded DNA and single-stranded RNA molecules, which bind to andform tertiary structures that specifically bind to a target molecule,such as a TβRII, TGFβ1, TGFβ2, and TGFβ3 polypeptide. The generation andtherapeutic use of aptamers are well established in the art. See, e.g.,U.S. Pat. No. 5,475,096. Additional information on aptamers can be foundin U.S. Patent Application Publication No. 20060148748. Nucleic acidaptamers are selected using methods known in the art, for example viathe Systematic Evolution of Ligands by Exponential Enrichment (SELEX)process. SELEX is a method for the in vitro evolution of nucleic acidmolecules with highly specific binding to target molecules as describedin, e.g., U.S. Pat. Nos. 5,475,096, 5,580,737, 5,567,588, 5,707,796,5,763,177, 6,011,577, and 6,699,843. Another screening method toidentify aptamers is described in U.S. Pat. No. 5,270,163. The SELEXprocess is based on the capacity of nucleic acids for forming a varietyof two- and three-dimensional structures, as well as the chemicalversatility available within the nucleotide monomers to act as ligands(form specific binding pairs) with virtually any chemical compound,whether monomeric or polymeric, including other nucleic acid moleculesand polypeptides. Molecules of any size or composition can serve astargets. The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievedesired binding affinity and selectivity. Starting from a mixture ofnucleic acids, which can comprise a segment of randomized sequence, theSELEX method includes steps of contacting the mixture with the targetunder conditions favorable for binding; partitioning unbound nucleicacids from those nucleic acids which have bound specifically to targetmolecules; dissociating the nucleic acid-target complexes; amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand enriched mixture of nucleic acids. The steps of binding,partitioning, dissociating and amplifying are repeated through as manycycles as desired to yield highly specific high affinity nucleic acidligands to the target molecule.

Typically, such binding molecules are separately administered to theanimal [see, e.g., O'Connor (1991) J. Neurochem. 56:560], but suchbinding molecules can also be expressed in vivo from polynucleotidestaken up by a host cell and expressed in vivo [see, e.g.,Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)].

3. Screening Assays

In certain aspects, the present invention relates to the use of TβRIIpolypeptides (e.g., soluble TβRII polypeptides) to identify compounds(agents) which are agonist or antagonists of the TGFβ1, TGFβ3 and TβRIIsignaling pathway. Compounds identified through this screening can betested to assess their ability to modulate TGFβ1 and TGFβ3 signalingactivity in vitro. Optionally, these compounds can further be tested inanimal models to assess their ability to modulate tissue growth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting TGFβ1, TGFβ3 and TβRIIpolypeptides. In certain embodiments, high-throughput screening ofcompounds can be carried out to identify agents that perturb TGFβ1,TGFβ3 or TβRII-mediated cell signaling. In certain embodiments, theassay is carried out to screen and identify compounds that specificallyinhibit or reduce binding of a TβRII polypeptide to TGFβ1 or TGFβ3.Alternatively, the assay can be used to identify compounds that enhancebinding of a TβRII polypeptide to TGFβ1 or TGFβ3. In a furtherembodiment, the compounds can be identified by their ability to interactwith a TGFβ1, TGFβ3 or TβRII polypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between a TβRIIpolypeptide and TGFβ1 or TGFβ3.

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified TβRII polypeptide which is ordinarily capable of binding toTGFβ1 or TGFβ3. To the mixture of the compound and TβRII polypeptide isthen added a composition containing a TβRII ligand. Detection andquantification of TβRII/TGFβ1 or TβRII/TGFβ3 complexes provides a meansfor determining the compound's efficacy at inhibiting (or potentiating)complex formation between the TβRII polypeptide and TGFβ1 or TGFβ3. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. For example, in a control assay, isolated and apurified TGFβ1 or TGFβ3 is added to a composition containing the TβRIIpolypeptide, and the formation of TβRII/TGFβ1 or TβRII/TGFβ3 complex isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the TβRII polypeptide and TGFβ1 or TGFβ3 maybe detected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeled TβRIIpolypeptide or TGFβ1 or TGFβ3, by immunoassay, or by chromatographicdetection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between a TβRII polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between a TβRII polypeptide andits binding protein. See for example, U.S. Pat. No. 5,283,317; Zervos etal. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between a TβRII polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with a TβRII or TGFβ1 or TGFβ3 polypeptide of theinvention. The interaction between the compound and the TβRII or TGFβ1or TGFβ3 polypeptide may be covalent or non-covalent. For example, suchinteraction can be identified at the protein level using in vitrobiochemical methods, including photo-crosslinking, radiolabeled ligandbinding, and affinity chromatography (Jakoby W B et al., 1974, Methodsin Enzymology 46: 1). In certain cases, the compounds may be screened ina mechanism based assay, such as an assay to detect compounds which bindto a TGFβ1 or TGFβ3 or TβRII polypeptide. This may include a solid-phaseor fluid-phase binding event. Alternatively, the gene encoding a TGFβ1or TGFβ3 or TβRII polypeptide can be transfected with a reporter system(e.g., 0-galactosidase, luciferase, or green fluorescent protein) into acell and screened against the library preferably by a high-throughputscreening or with individual members of the library. Othermechanism-based binding assays may be used, for example, binding assayswhich detect changes in free energy. Binding assays can be performedwith the target fixed to a well, bead or chip or captured by animmobilized antibody or resolved by capillary electrophoresis. The boundcompounds may be detected usually using colorimetric or fluorescence orsurface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor modulating (stimulating or inhibiting) TGFβ1- or TGFβ3-mediated cellsignaling. Therefore, any compound identified can be tested in wholecells or tissues, in vitro or in vivo, to confirm their ability tomodulate TGFβ1 or TGFβ3 signaling. Various methods known in the art canbe utilized for this purpose.

4. Exemplary Therapeutic Uses

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

The terms “treatment”, “treating”, “alleviation” and the like are usedherein to generally mean obtaining a desired pharmacologic and/orphysiologic effect, and may also be used to refer to improving,alleviating, and/or decreasing the severity of one or more symptoms of acondition being treated. The effect may be prophylactic in terms ofcompletely or partially delaying the onset or recurrence of a disease,condition, or symptoms thereof, and/or may be therapeutic in terms of apartial or complete cure for a disease or condition and/or adverseeffect attributable to the disease or condition. “Treatment” as usedherein covers any treatment of a disease or condition of a mammal,particularly a human, and includes: (a) preventing the disease orcondition from occurring in a subject which may be predisposed to thedisease or condition but has not yet been diagnosed as having it; (b)inhibiting the disease or condition (e.g., arresting its development);or (c) relieving the disease or condition (e.g., causing regression ofthe disease or condition, providing improvement in one or moresymptoms).

The terms “patient”, “subject”, or “individual” are used interchangeablyherein and refer to either a human or a non-human animal. These termsinclude mammals, such as humans, non-human primates, laboratory animals,livestock animals (including bovines, porcines, camels, etc.), companionanimals (e.g., canines, felines, other domesticated animals, etc.) androdents (e.g., mice and rats). In particular embodiments, the patient,subject or individual is a human.

As used herein, “combination”, “in combination with”, “conjointadministration” and the like refers to any form of administration suchthat the second therapy is still effective in the body (e.g., the twocompounds are simultaneously effective in the patient, which may includesynergistic effects of the two compounds). Effectiveness may notcorrelate to measurable concentration of the agent in blood, serum, orplasma. For example, the different therapeutic compounds can beadministered either in the same formulation or in separate formulations,either concomitantly or sequentially, and on different schedules. Thus,an individual who receives such treatment can benefit from a combinedeffect of different therapies. One or more TGFβ antagonists of thedisclosure can be administered concurrently with, prior to, orsubsequent to, one or more other additional agents or supportivetherapies. In general, each therapeutic agent will be administered at adose and/or on a time schedule determined for that particular agent. Theparticular combination to employ in a regimen will take into accountcompatibility of the antagonist of the present disclosure with thetherapy and/or the desired therapeutic effect to be achieved.

In part, the disclosure provides methods of treating heterotopicossification by administering to a patient in need thereof an effectiveamount of a TGFβ antagonists. In some embodiments, the methods relate topreventing or reducing the severity and/or duration of heterotopicossification in a patient in need thereof an effective amount of a TGFβantagonists. In some embodiments, the disclosure provides methods oftreating a disease or condition associated with heterotopic ossificationby administering to a patient in need thereof an effective amount of aTGFβ antagonists. In some embodiments, the methods relate to preventingor reducing the severity and/or duration of a disease or conditionassociated heterotopic ossification in a patient in need thereof aneffective amount of a TGFβ antagonists. Optionally, such methods furtherinclude administering the TGFβ antagonists in combination with one ormore additional active agents or supportive therapies for treatingheterotopic ossification.

In general, heterotopic ossification (HO) result from osteoid formationof mature lamellar bone in soft tissue sites outside the skeletalperiosteum (skeletal system). Often, there is an inciting event prior toonset of HO, usually an episode of trauma which may result in hematoma.In general, HO progression requires a supply of pluripotent(multipotent) mesenchymal cells, which can differentiate intoosteoblasts or chondroblasts. However, the mechanism of action behind HOdevelopment otherwise is largely unknown. Osteoid formation often isassociated with an inflammatory phase characterized by local swelling,pain, erythema and sometimes fever. This pathological process may occurin sites such as the skin, subcutaneous tissue, skeletal muscle, andfibrous tissue adjacent to joints. Bone may also form in walls of bloodvessels as well as in ligaments. Lesions range from small clinicallyinsignificant foci to massive deposits throughout the body. HO mayresult in joint contracture and ankylosis, pain, spasticity, swellingfever, neurovascular compression, pressure ulcers, and significantdisability. In some embodiments, the disclosure relates to methods oftreating one or more complication associated with heterotopicossification (e.g., osseous deposition, marrow space formation, jointcontracture, ankylosis, pain, spasticity, swelling, fever, neurovascularcompression, and pressure ulcers) by administering to a subject in needthereof an effective amount of a TGFβ antagonists, optionally incombination with one or more additional active agents or supportivetherapies for treating heterotopic ossification.

HO is most commonly it is associated with spinal cord injury, trauma,brain injuries, burns, fractures, muscle contusion, and jointarthroplasty. In some embodiments, the disclosure relates to methods oftreating heterotopic ossification associated with one or more disordersor conditions selected from the group consisting of: spinal cord injury,trauma, brain injuries, burns, fractures, muscle contusion, and jointarthroplasty by administering to a subject in need thereof an effectiveamount of a TGFβ antagonists, optionally in combination with one or moreadditional active agents or supportive therapies for treatingheterotopic ossification.

HO is a severe complication of hip, acetabular, and elbow fracturesurgery. About every third patient who has total hip arthroplasty (jointreplacement) or a severe fracture of the long bones of the lower legwill develop HO, but is uncommonly symptomatic. Between 50% and 90% ofpatients who developed HO following a previous hip arthroplasty willdevelop additional heterotopic ossification. In some embodiments, thedisclosure relates to methods of treating heterotopic ossificationassociated with one or more of joint, hip, acetabular, and elbowfracture surgery (e.g., replacement surgery) by administering to asubject in need thereof an effective amount of a TGFβ antagonists,optionally in combination with one or more additional active agents orsupportive therapies for treating heterotopic ossification.

HO often develops in patients with traumatic injury. In particular, HOis associated with brain or spinal cord injuries, which may account forthe clinical observation that traumatic brain injuries cause acceleratedfracture healing. Morley et al. (2005) Injury 36(3): 363. HO is alsoassociated with severe burns, combat-related trauma, and amputation. Intraumatic heterotopic ossification, the patient may complain of a warm,tender, firm swelling in a muscle and decreased range of motion in thejoint served by the muscle involved. There is often a history of a blowor other trauma to the area a few weeks to a few months earlier.Patients with traumatic neurological injuries, severe neurologicdisorders or severe burns who develop heterotopic ossificationexperience limitation of motion in the areas affected. In someembodiments, the disclosure relates to methods of treating heterotopicossification associated with one or more of traumatic injury, brain orspinal cord injuries, burns, combat-related trauma, and amputation byadministering to a subject in need thereof an effective amount of a TGFβantagonists, optionally in combination with one or more additionalactive agents or supportive therapies for treating heterotopicossification.

There are also rare genetic disorders causing heterotopic ossificationsuch as fibrodysplasia ossificans progressiva (FOP). FOP is an extremelyrare connective tissue disease. The disease is caused by a mutation ofthe body's repair mechanism, which causes fibrous tissue (includingmuscle, tendon, and ligament) to be ossified spontaneously or whendamaged. In many cases, injuries can cause joints to become permanentlyfrozen in place. For unknown reasons, children born with FOP havedeformed big toes, possibly missing a joint or simply presenting with anotable lump at the minor joint. The first “flare-up” that leads to theformation of FOP bones usually occurs before the age of 10. The bonegrowth progresses from the top downward, just as bones grow in fetuses.A child with FOP will typically develop bones starting at the neck, thenon the shoulders, arms, chest area and finally on the feet.Specifically, ossification is typically first seen in the dorsal, axial,cranial and proximal regions of the body. Later the disease progressesin the ventral, appendicular, caudal and distal regions of the body.However, it does not necessarily occur in this order due toinjury-caused flare-ups. Often, the tumor-like lumps that characterizethe disease appear suddenly. This condition causes loss of mobility toaffected joints, including inability to fully open the mouth limitingspeech and eating. Extra bone formation around the rib cage restrictsthe expansion of lungs and diaphragm causing breathing complications.Since the disease is so rare, the symptoms are often misdiagnosed ascancer or fibrosis. This leads physicians to order biopsies, which canexacerbate the growth of these lumps. The median age of survival is 40years with proper management. However, delayed diagnosis, trauma andinfections can decrease life expectancy. In general, FOP is caused by anautosomal dominant allele on chromosome 2q23-24. The allele has variableexpressivity, but complete penetrance. Most cases are caused byspontaneous mutation in the gametes. A similar but less catastrophicdisease is fibrous dysplasia, which is caused by a post-zygoticmutation. A mutation in the gene ACVR1 (also known as activin-likekinase 2 (ALK2)) is responsible for the disease. ACVR1 encodes activinreceptor type-1, a BMP type-1 receptor. The mutation causes substitutionof codon 206 from arginine to histidine in the ACVR1 protein. Thissubstitution causes abnormal activation of ACVR1, leading to thetransformation of connective tissue and muscle tissue into a secondaryskeleton. This causes endothelial cells to transform to mesenchymal stemcells and then to bone. In some embodiments, the disclosure relates tomethods of treating heterotopic ossification associated withfibrodysplasia ossificans progressiva by administering to a subject inneed thereof an effective amount of a TGFβ antagonists, optionally incombination with one or more additional active agents or supportivetherapies for treating heterotopic ossification and/or fibrodysplasiaossificans progressiva. In some embodiments, the disclosure relates tomethods of treating heterotopic ossification associated with fibrousdysplasia by administering to a subject in need thereof an effectiveamount of a TGFβ antagonists, optionally in combination with one or moreadditional active agents or supportive therapies for treatingheterotopic ossification and/or fibrous dysplasia.

There is no cure or approved treatment for FOP. Attempts to surgicallyremove the bone result in explosive bone growth. Clinical trials ofisotretinoin, etidronate with oral corticosteroids, and perhexilinemaleate have failed to demonstrate effectiveness, though the variablecourse of the disease and small prevalence induces uncertainty. Ahandful of pharmaceutical companies focused on rare disease arecurrently in varying stages of investigation into different therapeuticapproaches for FOP. In August 2015, U.S. Food and Drug AdministrationOffice of Orphan Products Development granted La Jolla Pharmaceuticalsorphan drug designation for two novel compounds for FOP. The compoundsare small-molecule kinase inhibitors designed to selectively block ACVR1(ALK2). In August 2015, Clementia Pharmaceuticals also began theenrollment of children into its Phase II clinical trial investigatingpalovarotene for the treatment of FOP. Preclinical studies demonstratedthat palovarotene, a retinoic acid receptor gamma agonist, blockedabnormal bone formation in animal models via inhibition of secondarymessenger systems in the BMP pathway. In September 2015, Regeneronannounced new insight into the mechanism of disease involving theactivation of the ACVR1 receptor by activin A. In 2016, the companyinitiated a phase 1 study of REGN 2477 (an activin A antibody) inhealthy volunteers. Another potential therapeutic approach involvesallele-specific RNA interference that targets mutated mRNA fordegradation while preserving normal ACVR1 gene expression. J. W. Loweryet al. (2012) Gene Therapy 19(701-702): 701-702. A mouse model of FOPexpressing a strong constitutively active ALK2 R206H mutant, was foundto be useful in identifying a selective agonist to nuclear retinoic acidreceptor-α (RAR-α) in mesenchymal cells. RAR-α agonists were found topartly inhibit HO, while an agonist to RAR-γ was found to be a potentinhibitor of intramuscular and subcutaneous HO in FOP models. Shimono etal., 2011, Nature Medicine 17:454-60). In some embodiments, thedisclosure relates to methods of treating heterotopic ossificationassociated with FOP by administering to a subject in need thereof aneffective amount of a TGFβ antagonists in combination with one or moreadditional active agents or supportive therapies for treating FOP,wherein the additional active agent or supportive therapy is selectedfrom the group consisting of: isotretinoin, etidronate with oralcorticosteroids, perhexiline maleate, ALK2 small-molecule inhibitors,palovarotene, retinoic acid receptor gamma agonists, retinoic acidreceptor alpha agonists, activin antibodies (e.g., activin A antibodiessuch as REGN 2477, and allele-specific RNA interference of ALK2.

Another rare genetic disorder causing heterotopic ossification isprogressive osseous heteroplasia (POH), which is a conditioncharacterized by cutaneous or subcutaneous ossification. POH isassociated with inactivating mutation in the GNAS gene, which encodesGas, the alpha subunit of the stimulatory guanine nucleotide bindingprotein that acts downstream of many G protein-coupled receptors inactivating adenylyl cyclase. Kaplan, et al. 1994, J Bone Joint Surg Am76, 425-436; Shore, et al., 2002, N Engl J Med 346, 99-106; and Eddy, etal., 2000, J Bone Miner Res 15, 2074-2083. Clinically, POH presentsduring infancy with dermal and subcutaneous ossifications that progressduring childhood into skeletal muscle and deep connective tissues (e.g.,tendon, ligaments, fascia). Over time these ossifications lead to jointstiffness, bone and joint fusions and growth retardation of the affectedlimbs. Currently, patients with POH undergo aggressive surgicalresection of ectopic bone to abrogate spreading of the lesion. Thisoften results in partial or full amputation of limbs and lesionsfrequently return (Kaplan, et al., 2000, J Bone Miner Res 15, 2084-94;and Shehab, et al., 2003, J Nucl Med 43, 346-353), which underscores theimportance of developing improved therapeutic interventions.Observations made in patients with POH suggest that mesenchymal stemcells present in soft tissues inappropriately differentiate intoosteoblasts and begin to deposit bone. Due to a lack of both in vitroand in vivo animal models, the pathogenesis of POH remains unknown and,like all forms of HO, lacks adequate treatments. In some embodiments,the disclosure relates to methods of treating heterotopic ossificationassociated with progressive osseous heteroplasia by administering to asubject in need thereof an effective amount of a TGFβ antagonists,optionally in combination with one or more additional active agents orsupportive therapies for treating heterotopic ossification and/orprogressive osseous heteroplasia.

HO also may occur in patients who are on neuromuscular blockade tomanage adult respiratory distress syndrome and in patients withnontraumatic myelopathies. In some embodiments, the disclosure relatesto methods of treating heterotopic ossification associated withneuromuscular blockade used manage adult respiratory distress syndromeby administering to a subject in need thereof an effective amount of aTGFβ antagonists, optionally in combination with one or more additionalactive agents or supportive therapies for treating heterotopicossification. In some embodiments, the disclosure relates to methods oftreating heterotopic ossification associated with nontraumaticmyelopathies by administering to a subject in need thereof an effectiveamount of a TGFβ antagonists, optionally in combination with one or moreadditional active agents or supportive therapies for treatingheterotopic ossification.

There is no clear form of treatment for HO. Originally, bisphosphonateswere expected to be of value after hip surgery but there has been noconvincing evidence of benefit, despite having been usedprophylactically. Prophylactic radiation therapy for the prevention ofheterotopic ossification has been employed since the 1970s. A variety ofdoses and techniques have been used. Generally, radiation therapy shouldbe delivered as close as practical to the time of surgery. A dose of 7-8Gray in a single fraction within 24-48 hours of surgery has been usedsuccessfully. Treatment volumes include the peri-articular region, andcan be used for hip, knee, elbow, shoulder, jaw or in patients afterspinal cord trauma. Single dose radiation therapy has been found to bewell tolerated and is cost effective, without an increase in bleeding,infection or wound healing disturbances. Certain anti-inflammatoryagents, such as indomethacin, ibuprofen and aspirin, have shown someeffect in preventing recurrence of heterotopic ossification after totalhip replacement. Conservative treatments such as passive range of motionexercises or other mobilization techniques provided by physicaltherapists or occupational therapists may also assist in preventing HO.In some embodiments, the disclosure relates to methods of treatingheterotopic ossification by administering to a subject in need thereofan effective amount of a TGFβ antagonists in combination with one ormore additional active agents or supportive therapies for treatingheterotopic ossification, wherein the one or more additional activeagents or supportive therapies is selected from the group consisting of:bisphosphonates, radiation therapy anti-inflammatory agents (e.g.,indomethacin, ibuprofen and aspirin), and conservative treatments suchas passive range of motion exercises or other mobilization techniques.

5. Pharmaceutical Compositions

The therapeutic agents described herein (e.g., TβRII fusionpolypeptides) may be formulated into pharmaceutical compositions.Pharmaceutical compositions for use in accordance with the presentdisclosure may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Such formulationswill generally be substantially pyrogen-free, in compliance with mostregulatory requirements.

In certain embodiments, the therapeutic method of the disclosureincludes administering the composition systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this disclosure is in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the TGFβ signalingantagonist which may also optionally be included in the composition asdescribed above, may be administered simultaneously or sequentially withthe subject compounds (e.g., TβRII polypeptides) in the methodsdisclosed herein.

Typically, protein therapeutic agents disclosed herein will beadministered parentally, and particularly intravenously orsubcutaneously. Pharmaceutical compositions suitable for parenteraladministration may comprise one or more TGFβ antagonists in combinationwith one or more pharmaceutically acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

The compositions and formulations may, if desired, be presented in apack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site. In certain embodiments, compositionsof the present invention may include a matrix capable of delivering oneor more therapeutic compounds (e.g., TGFβ antagonists) to a targettissue site, providing a structure for the developing tissue andoptimally capable of being resorbed into the body. For example, thematrix may provide slow release of the TGFβ antagonists. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalcium phosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., TGFβ antagonists). Thevarious factors include, but are not limited to, the patient's age, sex,and diet, the severity disease, time of administration, and otherclinical factors. Optionally, the dosage may vary with the type ofmatrix used in the reconstitution and the types of compounds in thecomposition. The addition of other known growth factors to the finalcomposition, may also affect the dosage. Progress can be monitored byperiodic assessment of bone growth and/or repair, for example, X-rays(including DEXA), histomorphometric determinations, and tetracyclinelabeling.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of TGFβ antagonists. Such therapy wouldachieve its therapeutic effect by introduction of the TGFβ antagonistpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of TGFβ antagonist polynucleotide sequences canbe achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Preferred for therapeuticdelivery of TGFβ antagonist polynucleotide sequences is the use oftargeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the TGFβ antagonist polynucleotide. In a preferredembodiment, the vector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for TGFβ antagonist polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

The disclosure provides formulations that may be varied to include acidsand bases to adjust the pH; and buffering agents to keep the pH within anarrow range.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments of thepresent invention, and are not intended to limit the invention.

Example 1. Generation of Receptor Fusion Protein Variants TβRII ECDVariants

TβRII fusion proteins comprising a soluble extracellular portion ofhuman TβRII and a human Fc portion were generated. For each fusionprotein, a TβRII amino acid sequence having the amino acid sequence ofSEQ ID NO: 18 was fused to an IgG Fc portion having the amino acidsequence of SEQ ID NO: 49 by means of one of several different linkers.Each of the fusion proteins also included a TPA leader sequence havingthe amino acid sequence of SEQ ID NO: 23 (below).

(SEQ ID NO: 23) Tissue plasminogen activator (TPA):MDAMKRGLCCVLLLCGAVFVSP

An illustration summary of several of the constructs designed isprovided as FIG. 3. A table detailing the sequences for the differentconstructs tested in the Exemplification section is provided below:

Construct Construct Amino Acid Name Sequence Linker Sequence hTβRII-hFcSEQ ID TGGG NO: 9 (SEQ ID NO: 3) hTβRII SEQ ID TGGGGSGGGGS (G4S)2-NO: 15 (SEQ ID NO: 4) hFc hTβRII SEQ ID TGGGGSGGGGSGGGGS (G4S)3- NO: 11(SEQ ID NO: 5) hFc hTβRII SEQ ID TGGGGSGGGGSGGGGS (G4S)4- NO: 13 GGGGShFc (SEQ ID NO: 6) hTβRII SEQ ID TGGGPKSCDK extended NO: 17(SEQ ID NO: 7) hinge-hFc hTpRII SEQ ID TGGGGSGGGGSGGGGS (G4S)5- NO: 44GGGGSGGGGS hFc (SEQ ID NO: 25) hTβRII SEQ ID TGGGGSGGGGSGGGGS (G4S)6-NO: 45 GGGGSGGGGSGGGGS hFc (SEQ ID NO: 26)

The amino acid sequences for the construct components and each of theconstructs, along with the nucleic acid sequence used to express theseconstructs, are provided below.

TβRII Portion: Amino Acid Sequence (SEQ ID NO: 18)   1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH      PLRHINNDMI VTDNNGAVKF  51 PQLCKFCDVR FSTCDNQKSC MSNCSITSIC      EKPQEVCVAV WRKNDENITL 101 ETVCHDPKLP YHDFILEDAA SPKCIMKEKK      KPGETFFMCS CSSDECNDNI 151 IFSEEYNTSN PD Fc Portion: Amino Acid Sequence (SEQ ID NO: 49)   1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM      ISRTPEVTCV VVDVSHEDPE  51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV      VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP      PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG      SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK hTβRII-hFc: Nucleic Acid Sequence(SEQ ID NO: 8)    1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT     GTGCTGCTGC TGTGTGGAGC   51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC     ACCGCACGTT CAGAAGTCGG  101 ATGTGGAAAT GGAGGCCCAG AAAGATGAAA     TCATCTGCCC CAGCTGTAAT  151 AGGACTGCCC ATCCACTGAG ACATATTAAT     AACGACATGA TAGTCACTGA  201 CAACAACGGT GCAGTCAAGT TTCCACAACT     GTGTAAATTT TGTGATGTGA  251 GATTTTCCAC CTGTGACAAC CAGAAATCCT     GCATGAGCAA CTGCAGCATC  301 ACCTCCATCT GTGAGAAGCC ACAGGAAGTC     TGTGTGGCTG TATGGAGAAA  351 GAATGACGAG AACATAACAC TAGAGACAGT     TTGCCATGAC CCCAAGCTCC  401 CCTACCATGA CTTTATTCTG GAAGATGCTG     CTTCTCCAAA GTGCATTATG  451 AAGGAAAAAA AAAAGCCTGG TGAGACTTTC     TTCATGTGTT CCTGTAGCTC  501 TGATGAGTGC AATGACAACA TCATCTTCTC     AGAAGAATAT AACACCAGCA  551 ATCCTGACAC CGGTGGTGGA ACTCACACAT     GCCCACCGTG CCCAGCACCT  601 GAACTCCTGG GGGGACCGTC AGTCTTCCTC     TTCCCCCCAA AACCCAAGGA  651 CACCCTCATG ATCTCCCGGA CCCCTGAGGT     CACATGCGTG GTGGTGGACG  701 TGAGCCACGA AGACCCTGAG GTCAAGTTCA     ACTGGTACGT GGACGGCGTG  751 GAGGTGCATA ATGCCAAGAC AAAGCCGCGG     GAGGAGCAGT ACAACAGCAC  801 GTACCGTGTG GTCAGCGTCC TCACCGTCCT     GCACCAGGAC TGGCTGAATG  851 GCAAGGAGTA CAAGTGCAAG GTCTCCAACA     AAGCCCTCCC AGCCCCCATC  901 GAGAAAACCA TCTCCAAAGC CAAAGGGCAG     CCCCGAGAAC CACAGGTGTA  951 CACCCTGCCC CCATCCCGGG AGGAGATGAC     CAAGAACCAG GTCAGCCTGA 1001 CCTGCCTGGT CAAAGGCTTC TATCCCAGCG     ACATCGCCGT GGAGTGGGAG 1051 AGCAATGGGC AGCCGGAGAA CAACTACAAG     ACCACGCCTC CCGTGCTGGA 1101 CTCCGACGGC TCCTTCTTCC TCTATAGCAA     GCTCACCGTG GACAAGAGCA 1151 GGTGGCAGCA GGGGAACGTC TTCTCATGCT     CCGTGATGCA TGAGGCTCTG 1201 CACAACCACT ACACGCAGAA GAGCCTCTCC     CTGTCTCCGG GTAAATGA hTβRII-hFc: Amino Acid Sequence (SEQ ID NO: 9)   1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV      QKSDVEMEAQ KDEIICPSCN  51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF      CDVRFSTCDN QKSCMSNCSI 101 TSICEKPQEV CVAVWRKNDE NITLETVCHD      PKLPYHDFIL EDAASPKCIM 151 KEKKKPGETF FMCSCSSDEC NDNIIFSEEY      NTSNPDTGGG THTCPPCPAP 201 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV      VVDVSHEDPE VKFNWYVDGV 251 EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD      WLNGKEYKCK VSNKALPAPI 301 EKTISKAKGQ PREPQVYTLP PSREEMTKNQ      VSLTCLVKGF YPSDIAVEWE 351 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV      DKSRWQQGNV FSCSVMHEAL 401 HNHYTQKSLS LSPGK

For the animal experiments described below, a mouse version of SEQ IDNO: 9 was generated by fusing the mouse extracellular domain of TβRII,which corresponds to isoform A of the human TβRII, to mouse G1Fc via alinker domain (TGGG). This fusion protein is designated herein asmTβRII-mFc.

hTβRII (G4S)3-hFc: Nucleic Acid Sequence (SEQ ID NO: 10)   1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101 ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151 AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201 CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251 GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301 ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351 GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401 CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451 AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501 TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551 ATCCTGACAC CGGTGGTGGA GGAAGTGGTG GAGGTGGTTC TGGAGGTGGT 601 GGAAGTACTC ACACATGCCC ACCGTGCCCA GCACCTGAAC TCCTGGGGGG 651 ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 701 CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC 751 CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC 801 CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC CGTGTGGTCA 851 GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG 901 TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 951 CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT1001 CCCGGGAGGA GATGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA1051 GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC1101 GGAGAACAAC TACAAGACCA CGCCTCCCGT GCTGGACTCC GACGGCTCCT1151 TCTTCCTCTA TAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG1201 AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC1251 GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA ATGAhTβRII (G4S)3-hFc: Amino Acid Sequence (SEQ ID NO: 11)   1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN  51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101 TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151 KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSGGG 201 GSTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 251 PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK 301 CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK 351 GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 401 NVFSCSVMHE ALHNHYTQKS LSLSPGKhTβRII (G45)4-hFc: Nucleic Acid Sequence (SEQ ID NO: 12)   1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101 ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151 AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201 CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251 GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301 ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351 GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401 CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451 AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501 TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551 ATCCTGACAC CGGTGGTGGA GGTTCTGGAG GTGGAGGAAG TGGTGGAGGT 601 GGTTCTGGAG GTGGTGGAAG TACTCACACA TGCCCACCGT GCCCAGCACC 651 TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG 701 ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC 751 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT 801 GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA 851 CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT 901 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT 951 CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT1001 ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG1051 ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA1101 GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG1151 ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT GGACAAGAGC1201 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT1251 GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAATGAhTβRII (G45)4-hFc: Amino Acid Sequence (SEQ ID NO: 13)   1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN  51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101 TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151 KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSGGG 201 GSGGGGSTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 251 VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 301 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL 351 TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS 401 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKhTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequence(SEQ ID NO: 63)   1 GATIPPHVQK SDVEMEAQKD EIICPSCNRT AHPLRHINND MIVTDNNGAV  51 KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 101 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND 151 NIIFSEEYNT SNPDTGGGGS GGGGSGGGGS GGGGSTHTCP PCPAPELLGG 201 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA 251 KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS 301 KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP 351 ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT 401 QKSLSLSPGKhTβRII (G45)4-hFc: Amino Acid Sequence lacking leader sequenceand lacking glycine prior to h7-81311 portion (SEQ ID NO: 64)   1 ATIPPHVQKS DVEMEAQKDE IICPSCNRTA HPLRHINNDM IVTDNNGAVK  51 FPQLCKFCDV RFSTCDNQKS CMSNCSITSI CEKPQEVCVA VWRKNDENIT 101 LETVCHDPKL PYHDFILEDA ASPKCIMKEK KKPGETFFMC SCSSDECNDN 151 IIFSEEYNTS NPDTGGGGSG GGGSGGGGSG GGGSTHTCPP CPAPELLGGP 201 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK 251 TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK 301 AKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE 351 NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ 401 KSLSLSPGKhTβRII (G45)4-hFc: Amino Acid Sequence lacking leader sequenceand lacking glycine, alanine, and threonine prior to h7-81311 portion(SEQ ID NO: 65)   1 IPPHVQKSDV EMEAQKDEII CPSCNRTAHP LRHINNDMIV TDNNGAVKFP  51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE 101 TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII 151 FSEEYNTSNP DTGGGGSGGG GSGGGGSGGG GSTHTCPPCP APELLGGPSV 201 FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK 251 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 301 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN 351 YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS  401 LSLSPGKhTβRII (G45)4-hFc: Amino Acid Sequence lacking leader sequenceand lacking glycine, alanine, threonine, and isoleucine priorto h7-81311 portion (SEQ ID NO: 66)   1 PPHVQKSDVE MEAQKDEIIC PSCNRTAHPL RHINNDMIVT DNNGAVKFPQ  51 LCKFCDVRFS TCDNQKSCMS NCSITSICEK PQEVCVAVWR KNDENITLET 101 VCHDPKLPYH DFILEDAASP KCIMKEKKKP GETFFMCSCS SDECNDNIIF 151 SEEYNTSNPD TGGGGSGGGG SGGGGSGGGG STHTCPPCPA PELLGGPSVF 201 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 251 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 301 QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY 351 KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL  401 SLSPGKhTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequenceand lacking glycine, alanine, threonine, isoleucine, and prolineprior to hTβRII portion (SEQ ID NO: 67)   1 PHVQKSDVEM EAQKDEITCP SCNRTAHPLR HINNDMIVTD NNGAVKFPQL  51 CKFCDVRFST CDNQKSCMSN CSITSICEKP QEVCVAVWRK NDENITLETV 101 CHDPKLPYHD FILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNIIFS 151 EEYNTSNPDT GGGGSGGGGS GGGGSGGGGS THTCPPCPAP ELLGGPSVFL 201 FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR 251 EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 301 PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK 351 TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS  401 LSPGKhTβRII (G45)4-hFc: Amino Acid Sequence lacking leader sequenceand lacking glycine, alanine, threonine, isoleucine, proline,and proline prior to hTβRII portion (SEQ ID NO: 68)   1 HVQKSDVEME AQKDEITCPS CNRTAHPLRH INNDMIVTDN NGAVKFPQLC  51 KFCDVRFSTC DNQKSCMSNC SITSICEKPQ EVCVAVWRKN DENITLETVC 101 HDPKLPYHDF ILEDAASPKC IMKEKKKPGE TFFMCSCSSD ECNDNIIFSE 151 EYNTSNPDTG GGGSGGGGSG GGGSGGGGST HTCPPCPAPE LLGGPSVFLF 201 PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE 251 EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP 301 REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 351 TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL  401 SPGKhTβRII (G45)2-hFc: Nucleic Acid Sequence (SEQ ID NO: 14)   1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101 ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151 AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201 CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251 GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301 ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351 GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401 CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451 AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501 TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551 ATCCTGACAC CGGTGGAGGT GGTTCTGGAG GTGGTGGAAG TACTCACACA 601 TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT 651 CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG 701 TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC 751 AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG 801 GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC 851 TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC 901 AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA 951 GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA1001 CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC1051 GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA1101 GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTATAGCA1151 AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC1201 TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC1251 CCTGTCTCCG GGTAAATGA hTβRII (G4S)2-hFc: Amino Acid Sequence(SEQ ID NO: 15)   1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN  51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101 TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151 KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSTHT 201 CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 251 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN 301 KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS 351 DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC 401 SVMHEALHNH YTQKSLSLSP GKhTβRII extended hinge-hFc: Nucleic Acid Sequence (SEQ ID NO: 16)   1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101 ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151 AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201 CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251 GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301 ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351 GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401 CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451 AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501 TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551 ATCCTGACAC CGGTGGTGGA CCCAAATCTT GTGACAAAAC TCACACATGC 601 CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAG TCTTCCTCTT 651 CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA 701 CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC 751 TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA 801 GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC 851 ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA 901 GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC 951 CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA1001 AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC1051 ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC1101 CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TATAGCAAGC1151 TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC1201 GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT1251 GTCCCCGGGT AAATGA hTβRII extended hinge-hFc: Amino Acid Sequence(SEQ ID NO: 17)   1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN  51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101 TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151 KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG PKSCDKTHTC 201 PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN 251 WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK 301 ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD 351 IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS 401 VMHEALHNHY TQKSLSLSPG K hTβRII (G45)5-hFc: Amino Acid Sequence(SEQ ID NO: 44)   1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN  51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101 TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151 KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSGGG 201 GSGGGGSGGG GSTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT 251 CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH 301 QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK 351 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 401 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKhTβRII (G4S)6-hFc: Amino Acid Sequence (SEQ ID NO: 45)   1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEITCPSCN  51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101 TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151 KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSGGG 201 GSGGGGSGGG GSGGGGSTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR 251 TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 301 LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 351 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF 401 LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKhTβRII (G45)5-hFc: Nucleotide Sequence (SEQ ID NO: 46)   1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101 ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151 AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201 CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251 GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301 ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351 GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401 CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451 AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501 TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551 ATCCTGACAC CGGTGGAGGA GGTTCTGGTG GTGGAGGTTC TGGAGGTGGA 601 GGAAGTGGTG GAGGTGGTTC TGGAGGTGGT GGAAGTACTC ACACATGCCC 651 ACCGTGCCCA GCACCTGAAC TCCTGGGGGG ACCGTCAGTC TTCCTCTTCC 701 CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC TGAGGTCACA 751 TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG 801 GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG 851 AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC 901 CAGGACTGGC TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC 951 CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAGCCAAA GGGCAGCCCC1001 GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGAGGA GATGACCAAG1051 AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT1101 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA1151 CGCCTCCCGT GCTGGACTCC GACGGCTCCT TCTTCCTCTA TAGCAAGCTC1201 ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT1251 GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT1301 CTCCGGGTAA ATGA hTβRII (G45)6-hFc: Nucleotide Sequence(SEQ ID NO: 47)   1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101 ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151 AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201 CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251 GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301 ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351 GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401 CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451 AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501 TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551 ATCCTGACAC CGGTGGAGGT GGAAGTGGTG GAGGAGGTTC TGGTGGTGGA 601 GGTTCTGGAG GTGGAGGAAG TGGTGGAGGT GGTTCTGGAG GTGGTGGAAG 651 TACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT 701 CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG 751 ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA 801 GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA 851 CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC 901 CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA 951 GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG1001 CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG1051 GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT1101 CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA1151 ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC1201 CTCTATAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT1251 CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA1301 AGAGCCTCTC CCTGTCTCCG GGTAAATGA

The various constructs were successfully expressed in CHO cells and werepurified to a high degree of purity as determined by analyticalsize-exclusion chromatography and SDS-PAGE. The hTβRII (G4S)2-hFc,hTβRII (G4S)3-hFc, hTβRII (G4S)4-hFc, hTβRII (G4S)5-hFc and hTβRII(G4S)6-hFc proteins displayed similarly strong stability as determinedby SDS-PAGE analysis when maintained in PBS for 13 days at 37° C. ThehTβRII (G4S)2-hFc, hTβRII (G4S)3-hFc, hTβRII (G4S)4-hFc proteins werealso maintained in rat, mouse or human serum and displayed similarlystrong stability.

TβRII ECD Variants

In addition to the TβRII domains included in the fusion proteinsdescribed above (e.g., SEQ ID NO: 18), the disclosure also contemplatesfusion proteins comprising alternative TβRII domains. For example, thefusion protein may comprise the wild-type hTβRII_(short)(23-159)sequence shown below (SEQ ID NO: 27) or any of the other TβRIIpolypeptides disclosed below:

(SEQ ID NO: 27)   1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK    FCDVRFSTCD NQKSCMSNCS  51 ITSICEKPQE VCVAVWRKND ENITLETVCH    DPKLPYHDFI LEDAASPKCI 101 MKEKKKPGET FFMCSCSSDE CNDNIIFSEE    YNTSNPD(1) The hTβRII_(short)(23-159/D110K) amino acid sequence shown below(SEQ ID NO: 36), in which the substituted residue is underlined.

(SEQ ID NO: 36)   1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK    FCDVRFSTCD NQKSCMSNCS  51 ITSICEKPQE VCVAVWRKND ENITLETVCH    DPKLPYHKFI LEDAASPKCI 101 MKEKKKPGET FFMCSCSSDE CNDNIIFSEE    YNTSNPD(2) The N-terminally truncated hTβRII_(short)(29-159) amino acidsequence shown below (SEQ ID NO: 28).

(SEQ ID NO: 28)   1 QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF    STCDNQKSCM SNCSITSICE  51 KPQEVCVAVW RKNDENITLE TVCHDPKLPY    HDFILEDAAS PKCIMKEKKK 101 PGETFFMCSC SSDECNDNII FSEEYNTSNP     D(3) The N-terminally truncated hTβRII_(short)(35-159) amino acidsequence shown below (SEQ ID NO: 29).

(SEQ ID NO: 29)   1 DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ    KSCMSNCSIT SICEKPQEVC  51 VAVWRKNDEN ITLETVCHDP KLPYHDFILE    DAASPKCIMK EKKKPGETFF 101 MCSCSSDECN DNIIFSEEYN TSNPD(4) The C-terminally truncated hTβRII_(short)(23-153) amino acidsequence shown below (SEQ ID NO: 30).

(SEQ ID NO: 30)   1TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSNCS  51ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE Y(5) The C-terminally truncated hTβRII_(short)(23-153/N70D) amino acidsequence shown below (SEQ ID NO: 38), in which the substituted residueis underlined.

(SEQ ID NO: 38)   1TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSDCS  51ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE Y

Applicants also envision five corresponding variants (SEQ ID NOs: 37,33, 34, 39) based on the wild-type hTβRII_(long)(23-184) sequence shownabove and below (SEQ ID NO: 20), in which the 25 amino-acid insertion isunderlined. Note that splicing results in a conservative amino acidsubstitution (Val→Ile) at the flanking position C-terminal to theinsertion.

(SEQ ID NO: 20)   1TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF  51PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEYNTSN PD(1) The hTβRII_(long)(23-184/D135K) amino acid sequence shown below (SEQID NO: 37), in which the substituted residue is double underlined.

(SEQ ID NO: 37)   1TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF  51PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHKFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEYNTSN PD(2) The N-terminally truncated hTβRII_(long)(29-184) amino acid sequenceshown below (SEQ ID NO: 33).

(SEQ ID NO: 33)   1QKSDVEMEAQ KDEIICPSCN RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF  51CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD 101PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEEY 151 NTSNPD(3) The N-terminally truncated hTβRII_(long)(60-184) amino acid sequenceshown below (same as SEQ ID NO: 29).

(same as SEQ ID NO: 29)   1DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC  51VAVWRKNDEN ITLETVCHDP KLPYHDFILE DAASPKCIMK EKKKPGETFF 101MCSCSSDECN DNIIFSEEYN TSNPD(4) The C-terminally truncated hTβRI_(long)(23-178) amino acid sequenceshown below (SEQ ID NO: 34).

(SEQ ID NO: 34)   1TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF  51PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEY(5) The C-terminally truncated hTβRII_(long)(23-178/N95D) amino acidsequence shown below (SEQ ID NO: 39), in which the substituted residueis double underlined.

(SEQ ID NO: 39)   1TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF  51PQLCKFCDVR FSTCDNQKSC MSDCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEY

Additional TβRII ECD variants include:

(A) The N- and C-terminally truncated hTβRII_(short)(35-153) orhTβRII_(long)(60-178) amino acid sequence shown below (SEQ ID NO: 32).

(SEQ ID NO: 32)   1DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC  51VAVWRKNDEN ITLETVCHDP KLPYHDFILE DAASPKCIMK EKKKPGETFF 101MCSCSSDECN DNIIFSEEY(B) The N- and C-terminally truncated hTβRII_(short)(29-153) amino acidsequence shown below (SEQ ID NO: 31).

(SEQ ID NO: 31)   1QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF STCDNQKSCM SNCSITSICE  51KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS PKCIMKEKKK 101PGETFFMCSC SSDECNDNII FSEEY(C) The N- and C-terminally truncated hTβRII_(long)(29-178) amino acidsequence shown below (SEQ ID NO: 35).

(SEQ ID NO: 35)   1QKSDVEMEAQ KDEIICPSCN RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF  51CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD 101PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEEY

Any of the above variants (SEQ ID NO: 36, 28, 29, 30, 38, 37, 33, 34,39, 32, 31, and 35) could incorporate an insertion of 36 amino acids(SEQ ID NO: 41) between the pair of glutamate residues (positions 151and 152 of SEQ ID NO: 1, or positions 176 and 177 of SEQ ID NO: 2)located near the C-terminus of the hTβRII ECD, as occurs naturally inthe hTβRII isoform C (Konrad et al., BMC Genomics 8:318, 2007).

(SEQ ID NO: 41) GRCKIRHIGS NNRLQRSTCQ NTGWESAHVM KTPGFR

As an example, the paired glutamate residues flanking the optionalinsertion site are denoted below (underlined) for thehTβRII_(short)(29-159) variant (SEQ ID NO: 28).

(SEQ ID NO: 28)   1QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF STCDNQKSCM SNCSITSICE  51KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS PKCIMKEKKK 101PGETFFMCSC SSDECNDNII FSEEYNTSNP D

Fc Domain Variants

While the constructs described above were generated with an Fe domainhaving the amino acid sequence of SEQ ID NO: 49, the disclosurecontemplates hTβRII-hFc fusion proteins comprising alternative Fcdomains, including a human IgG2 Fc domain (SEQ ID NO: 42, below) orfull-length human IgG1 Fc (hG1Fc) (SEQ ID NO: 43, below). Optionally, apolypeptide unrelated to an Fc domain could be attached in place of theFc domain.

(SEQ ID NO: 42)   1VECPPCPAPP VAGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVQ  51FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS 101NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP 151SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS 201CSVMHEALHN HYTQKSLSLS PGK (SEQ ID NO: 43)   1GGPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV  51DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL 101NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS 151LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK 201SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

Leader Sequence Variants

While the generated constructs described above included the TPA leadersequence, alternative leader sequences may be used, such as the nativeleader sequence (SEQ ID NO: 22—below) or the honey bee melittin (SEQ IDNO: 24—below) leader sequences.

Native: MGRGLLRGLWPLHIVLWTRIAS (SEQ ID NO: 22)Honey bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 24)

Example 2. Differential Ligand Inhibition by Receptor Fusion ProteinVariants in Cell-Based Assay

Affinities of TGFβ1, TGFβ2 and TGFβ3 for hTβRII (G4S)2-hFc; hTβRII(G4S)3-hFc; hTβRII (G4S)4-hFc; hTβRII-hFc; and hTβRII extended hinge-hFcproteins were evaluated in vitro with a Biacore™ instrument, and theresults are summarized in FIGS. 4A and 4B. Each of the fusion proteinswas capable of binding TGFβ1 and TGFβ3 with high affinity, but theconstructs having linker lengths longer than or equal to (G4S)4 weresurprisingly capable of binding to both TGFβ1 and TGFβ3 with higheraffinity than constructs having linker lengths shorter than (G4S)4.Binding between TGFβ2 and any of the constructs was low or transient.Deglycosylation of the constructs did not change binding.

A reporter gene assay in A549 cells was used to determine the ability ofhTβRII-hFc variants to inhibit activity of TGFβ1, TGFβ2 and TGFβ3. Thisassay is based on a human lung carcinoma cell line transfected with apGL3(CAGA)12 reporter plasmid (Dennler et al, 1998, EMBO 17: 3091-3100)as well as a Renilla reporter plasmid (pRLCMV) to control fortransfection efficiency. The CAGA motif is present in the promoters ofTGFβ-responsive genes (for example, PAI-1), so this vector is of generaluse for factors signaling through SMAD2 and SMAD3.

On the first day of the assay, A549 cells (ATCC®: CCL-185™) weredistributed in 48-well plates. On the second day, a solution containingpGL3(CAGA)12, pRLCMV, X-tremeGENE 9 (Roche Applied Science), and OptiMEM(Invitrogen) was preincubated, then added to Eagle's minimum essentialmedium (EMEM, ATCC®) supplemented with 0.1% BSA, which was applied tothe plated cells for incubation overnight at 37° C., 5% CO₂. On thethird day, medium was removed, and cells were incubated overnight at 37°C., 5% CO₂ with a mixture of ligands and inhibitors prepared asdescribed below.

Serial dilutions of test articles were made in a 48-well plate in assaybuffer (EMEM+0.1% BSA). An equal volume of assay buffer containing thetest ligand was added to obtain a final ligand concentration equal tothe EC50 determined previously. Human TGFβ1, human TGFβ2, and humanTGFβ3 were obtained from PeproTech. Test solutions were incubated at 37°C. for 30 minutes, then a portion of the mixture was added to all wells.After incubation with test solutions overnight, cells were rinsed withphosphate-buffered saline, then lysed with passive lysis buffer (PromegaE1941) and stored overnight at −70° C. On the fourth and final day,plates were warmed to room temperature with gentle shaking. Cell lysateswere transferred in duplicate to a chemiluminescence plate (96-well) andanalyzed in a luminometer with reagents from a Dual-Luciferase ReporterAssay system (Promega E1980) to determine normalized luciferaseactivity.

As illustrated in FIGS. 5A-5F, the hTβRII (G4S)2-hFc; hTβRII (G4S)3-hFc;hTβRII (G4S)4-hFc; hTβRII (G4S)5-hFc; hTβRII (G4S)6-hFc; hTβRII-hFc; andhTβRII extended hinge-hFc proteins all were capable of inhibiting bothTGFβ1 and TGFβ3. Interestingly, while there was a correlation betweenimproved TGFβ1 and TGFβ3 inhibition and linker length for the hTβRII(G4S)2-hFc; hTβRII (G4S)3-hFc and hTβRII (G4S)4-hFc constructs (FIG.5E), this improvement trend appeared to have plateaued for hTβRII(G4S)5-hFc and hTβRII (G4S)6-hFc constructs (FIG. 5F).

Example 3. Effects of TβRII-Fc in a Heterotopic Ossification AnimalModel

Heterotopic ossification (HO) can commonly occur after severe trauma,burn injuries, and is a debilitating consequence of the congenitaldisease fibrodysplasia ossificans progressive (FOP). The etiologyremains poorly understood, however, it is presumed that inflammationplays a critical role with several inflammatory cell types beingrecruited to the site of HO development. The effects of mTβRII-mFc, asdescribed above, on the progression of HO were investigated using a HOanimal model.

Recruited macrophages are a known source of cytokines that influence theinflammatory microenvironment. Therefore, the secretion of TGFβ1 incultured macrophages was initially assessed. Bone marrow derivedmacrophages were isolated and polarized into M2 phenotype in-vitro.Secreted TGFβ was measured in conditioned media using ELISA.Interestingly, it was observed that, while MO macrophages secrete onlyminimal TGFβ1, the regenerative M2 macrophages, which are known to berecruited to inflammatory sites such as those occurring at sites ofosseous deposition in HO, had a 500-fold increase in TGFβ1 secretion.Furthermore, the increase in TGFβ1 secretion was completely abrogatedwhen the mTβRII-mFc (5.44 pg/ml) was present in the culture media (FIG.6A).

Next, the effect of systemically administered mTβRII-mFc were observedin a model of traumatic heterotopic ossification involving a 30% dorsalburn and Achilles tenotomy. Agarwal et al. (2016) Proc Natl Acad Sci USA113(3): E338-347. Six-week old male C57BL/6 mice were induced for HO andthen randomized into one of two treatment groups: 1) treatment with themTβRII-mFc (10 mg/kg) (n=10) or 2) PBS control (n=10). This wasadministered subcutaneously twice weekly for 3 weeks. At 3 weeks,histology samples were collected, decalcified and stained with SafraninO to assess formation of HO anlagen. Volume of mature formed HO wasquantified using micro CT analysis and imaging reconstruction at 9weeks. Following treatment for 3 weeks, a substantially attenuateddevelopment of HO anlagen in mice treated with mTβRII-mFc was observedupon histologic examination along with decreased osseous deposition andmarrow space formation (FIG. 6B). Furthermore, the volume of mature HOwas significantly decreased in the mTβRII-mFc treatment group (FIG. 6C).

In this study, it was demonstrated that TGFβ1 signaling plays a criticalrole in HO formation and treatment with mTβRII-mFc is effective inattenuating ectopic bone deposition, thus resulting in decreased HOvolume. Therefore, these data suggest that TβRII polypeptides, as wellas other TGFβ antagonist, may be used to treat HO, revealing a newavenue for the treatment of this condition in human patients.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1-84. (canceled)
 85. A method for treating heterotopic ossification (HO)in a patient in need thereof, comprising administering to the patient aneffective amount of a Transforming Growth Factor-β (TGFβ) antagonist,wherein the TGFβ antagonist is a Transforming Growth Factor-β ReceptorII (TβRII) polypeptide.
 86. The method of claim 85, wherein theheterotopic ossification is associated with one or more disorders orconditions selected from the group consisting of: spinal cord injury,trauma, brain injuries, burns, fractures, muscle contusion, jointarthroplasty/replacement, hip surgery/replacement, acetabularsurgery/replacement, elbow fracture, fracture of the long bones of thelower leg, combat-related trauma, amputation, neuromuscular blockadeused to manage adult respiratory distress syndrome, non-traumaticmyelopathies.
 87. The method of claim 85, wherein the compositionprevents or reduces the severity or duration of heterotopic ossificationin the patient or one or more complications of heterotopic ossificationselected from the group consisting of: joint contracture, ankylosis,pain, spasticity, swelling fever, neurovascular compression, andpressure ulcers.
 88. The method of claim 85, wherein the heterotopicossification occurs in one or more tissues selected from the groupconsisting of: bone, skin, subcutaneous tissue, skeletal muscle,fibrosis tissue adjacent to joints, walls of blood vessels, andligaments.
 89. The method of claim 85, wherein the composition isadministered in combination with an additional active agent orsupportive therapy for treating heterotopic ossification, FOP, POH, orfibrous dysplasia, said additional agent or supportive therapy beingselected from the group consisting of: isotretinoin, etidronate withoral corticosteroids, perhexiline maleate, ALK2 small-moleculeinhibitors, palovarotene, retinoic acid receptor gamma agonists,retinoic acid receptor alpha agonists, activin antibodies,allele-specific RNA interference of ALK2, bisphosphonates, radiationtherapy anti-inflammatory agents, or conservative treatments such aspassive range of motion exercises or other mobilization techniques. 90.The method of claim 85, wherein the TβRII polypeptide comprises an aminoacid sequence selected from: a. an amino acid sequence at least 75%,90%, 95%, 99%, or 100% identical to a sequence beginning at any one ofpositions 23 to 35 of SEQ ID NO: 1 and ending at any one of positions153 to 159 of SEQ ID NO: 1; and b. an amino acid sequence at least 75%,90%, 95%, 99%, or 100% identical to a sequence beginning at any one ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any one of positions178 to 184 of SEQ ID NO:
 2. 91. The method of claim 85, wherein theTβRII polypeptide comprises an amino acid sequence at least 80%, 90%, or100% identical to SEQ ID NO:
 18. 92. The method of claim 85, wherein theTβRII polypeptide is a fusion polypeptide comprising: a. anextracellular domain of a TβRII portion; and b. a heterologous portion,and wherein the TβRII extracellular domain portion comprises an aminoacid sequence at least 80% identical to: i) a sequence beginning at anyof positions 23 to 35 of SEQ ID NO: 1 and ending at any of positions 153to 159 of SEQ ID NO: 1 or ii) a sequence beginning at any of positions23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to 184 ofSEQ ID NO:
 2. 93. The method of claim 92, wherein the TβRII polypeptidecomprises an amino acid sequence at least 80% identical to SEQ ID NO:18, or the TβRII extracellular domain portion consists of an amino acidsequence at least 80% identical to SEQ ID NO:
 18. 94. The method ofclaim 92 or 93, wherein the heterologous portion is an immunoglobulin Fcdomain.
 95. The method of claim 94, wherein the heterologous portioncomprises an amino acid sequence that is at least 80% or 100% identicalto SEQ ID NO:
 49. 96. The method of claim 92, wherein the fusion proteinfurther comprises a linker domain.
 97. The method of claim 96, whereinthe linker: (a) is between 10 and 25 amino acids in length; or (b)comprises an amino acid sequence selected from: a. (GGGGS)n, whereinn=>2; b. (GGGGS)n, wherein n=>3; c. (GGGGS)n, wherein n=>4; and theamino acid sequence of any one of SEQ ID Nos: 4-7, 19, 21, 25, 26, and40; or (c) comprises (GGGGS)n, wherein n>5.
 98. The method of claim 85,wherein the TβRII polypeptide comprises an amino acid sequence that isat least 80% identical to the amino acid sequence of SEQ ID NOs: 11, 13,15, 65, or
 68. 99. The method of claim 85, wherein: (1) the TβRIIpolypeptide consists of: a) a TβRII polypeptide portion comprising anamino acid sequence that is at least 85%, 90%, 95%, 97%, 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 18 and no more than10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linkerportion comprising an amino acid sequence that is at least 85%, 90%,95%, 97%, 99%, or 100% identical to the amino acid sequence of SEQ IDNO: 6 and no more than 5, 4, 3, 2 or 1 additional amino acids; c) aheterologous portion comprising an amino acid sequence that is at least85%, 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence ofSEQ ID NO: 49 and no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1additional amino acids; and d) optionally a leader sequence (e.g., SEQID NO: 23); or (2) the TβRII polypeptide comprises: a) an extracellulardomain of a TβRII portion; wherein the extracellular domain comprises anamino acid sequence that is at least 85%, 90%, 95%, 97%, 99%, or 100%identical to the sequence of SEQ ID NO: 18; b) a heterologous portion,wherein the heterologous portion comprises an amino acid sequence thatis at least 85%, 90%, 95%, 97%, 99%, or 100% identical to the sequenceof SEQ ID NO: 49; and c) a linker portion connecting the extracellulardomain and the heterologous portion; wherein the linker comprises anamino acid sequence that is at least 85%, 90%, 95%, 97%, 99%, or 100%identical to the amino acid sequence of SEQ ID NO:
 6. 100. The method ofclaim 85, wherein the TβRII polypeptide comprises an amino acid sequencethat is at least 85%, 90%, 95%, 97%, 99%, or 100% identical to the aminoacid sequence of SEQ ID NO:
 48. 101. The method of claim 85, wherein theTβRII polypeptide or fusion polypeptide includes one or more modifiedamino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, and an amino acid conjugated to a lipidmoiety.
 102. The method of claim 85, wherein the TβRII polypeptide orfusion polypeptide: (1) binds to TGFβ1 and TGFβ3, and/or (2) inhibitsTGFβ1 and TGFβ3 signaling as determined using a reporter gene assay.103. The method of claim 85, wherein the TβRII polypeptide or fusionpolypeptide is a homodimer.